(n-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide

ABSTRACT

The present invention relates to stable novel salt forms of N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide that are suitable for the preparation of pharmaceutical formulations thereof, and their therapeutic use.

This application is a continuation of U.S. patent application Ser. No.14/736,690, filed Jun. 11, 2015, which claims the benefit of U.S. PatentApplication Ser. No. 62/011,315, filed Jun. 12, 2014, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD

The present application relates to stable novel salt forms ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidethat are suitable for the preparation of pharmaceutical formulationsthereof, and their therapeutic use.

BACKGROUND

The inhibition to Janus kinase (JAK) has been evaluated in treatinghyperproliferative diseases. Several JAK inhibitors have been developed:ruxolitinib, tofacitinib, baricitinib, lestaurtinib, pacritinib,fedratinib, XL019, SB 1518 and AZD1480 have been developed (Sonbol,Ther. Adv. Hematol. 4: 15-35, 2013). The compoundN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) is a JAK kinase inhibitor

In clinical studies, CYT-0387 is effective in treatinghyperproliferative diseases such as polycythemia vera (PV), essentialthrombocythemia (ET), and primary myelofibrosis (PMF). Also, thepatients having myelofibrosis who received CYT-0387 exhibited theimprovement in the anemia and/or spleen responses (see U.S. Pat. No.8,486,941 and Application Publication No. 2014-0073643, each of which isincorporated herein by reference in its entirety).

It is desired to have different forms of the compound that are suitablefor the preparation of pharmaceutical formulations containing CYT-0387and their therapeutic use.

BRIEF SUMMARY

The present invention is directed to novel CYT-0387 forms.

In one aspect, the present invention is directed to CYT-0387monohydrochloride anhydrous Form I:

which has an X-ray powder diffraction (XRPD) pattern having peaks atabout 13.5°, 20.9°, 26.1°, 26.6°, and 28.3° 2-θ±0.2° 2-θ.

In another aspect, the present invention is directed to CYT-0387dihydrochloride monohydrate Form II:

which has an X-ray powder diffraction (XRPD) pattern having peaks atabout 7.7°, 19.3°, 24.0°, 25.7°, and 29.6° 2-θ±0.2° 2-θ.

In another aspect, the present invention is directed to CYT-0387monohydrochloride anhydrous Form III (Form III):

which has an X-ray powder diffraction (XRPD) pattern having peaks atabout 12.7°, 14.6°, 17.8°, 19.7°, and 23.3° 2-θ±0.2° 2-θ.

The invention also provides compositions, including pharmaceuticalcompositions, kits that include the compounds, and methods of using andmaking the pharmaceutical compositions. The pharmaceutical compositionsprovided herein are useful in treating diseases, disorders, orconditions that are mediated by JAK.

In one embodiment, the application is directed to dosage formscomprising CYT-0387 Form III, in particular dosage forms as a tablet,and more particularly dosage forms comprising comprises CYT-0387 FormIII in an amount equivalent to from between 30-250 mg of CYT-0387 freebase. In one embodiment, the dosage form comprises CYT-0387 Form II inan amount equivalent to 100-200 mg of CYT-0387 free base.

In further embodiments, the application is directed to dosage forms orpharmaceutical compositions comprising CYT-0387 Form III in an amountequivalent to 200 mg of CYT-0387 free base which provide apharmacokinetic profile substantially similar to a dosage form orpharmaceutical composition comprising the CYT-0387 dihydrochlorideanhydrous Form I in an amount equivalent to 300 mg of CYT-0387 freebase.

In some embodiments, the application is directed to dosage formscomprising CYT-0387 dihydrochloride monohydrate Form II, as a tablet,wherein the dosage forms comprising comprises CYT-0387 dihydrochloridemonohydrate Form II in an amount equivalent to from between about 100 mgto about 300 mg of CYT-0387 free base. In certain embodiments, theapplication is directed to dosage forms comprising CYT-0387dihydrochloride monohydrate Form II, as a tablet, wherein the dosageforms comprising comprises CYT-0387 dihydrochloride monohydrate Form IIin an amount equivalent to about 100 mg, about 150 mg, about 200 mg,about 250 mg, or about 300 mg of CYT-0387 free base. In certainembodiments, the application is directed to dosage forms comprisingCYT-0387 dihydrochloride monohydrate Form II, as a tablet, wherein thedosage forms comprising comprises CYT-0387 dihydrochloride monohydrateForm II in an amount equivalent to about 100 mg of CYT-0387 free base.In certain embodiments, the application is directed to dosage formscomprising CYT-0387 dihydrochloride monohydrate Form II, as a tablet,wherein the dosage forms comprising comprises CYT-0387 dihydrochloridemonohydrate Form II in an amount equivalent to about 200 mg of CYT-0387free base. In certain embodiments, the application is directed to dosageforms comprising CYT-0387 dihydrochloride monohydrate Form II, as atablet, wherein the dosage forms comprising comprises CYT-0387dihydrochloride monohydrate Form II in an amount equivalent to about 250mg of CYT-0387 free base. In certain embodiments, the application isdirected to dosage forms comprising CYT-0387 dihydrochloride monohydrateForm II, as a tablet, wherein the dosage forms comprising comprisesCYT-0387 dihydrochloride monohydrate Form II in an amount equivalent toabout 300 mg of CYT-0387 free base. In additional embodiments, theapplication is directed to dosage forms or pharmaceutical compositionscomprising CYT-0387 dihydrochloride monohydrate Form II in an amountequivalent to 200 mg of CYT-0387 free base which provide apharmacokinetic profile substantially similar to a dosage form orpharmaceutical composition comprising the CYT-0387 dihydrochloridemonohydrate Form II in an amount equivalent to 300 mg of CYT-0387 freebase.

Also provided is a kit that includes a compound of formula (I) or apharmaceutically acceptable salt, isomer, or a mixture thereof. The kitmay further comprise a label and/or instructions for use of the compoundin treating a disease, disorder, or condition in a human in needthereof.

Also provided are articles of manufacture that include a compound offormula (I) or a pharmaceutically acceptable salt, isomer, or a mixturethereof, and a container. In one embodiment, the container may be avial, jar, ampoule, preloaded syringe, or an intravenous bag.

DESCRIPTION OF THE FIGURES

FIG. 1: XRPD ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride anhydrous Form I.

FIG. 2: DSC ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride anhydrous Form I.

FIG. 3: TGA ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride anhydrous Form I.

FIG. 4: DVS ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride anhydrous Form I.

FIG. 5: XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II.

FIG. 6: XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I.

FIG. 7: XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III.

FIG. 8: DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II.

FIG. 9: DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I.

FIG. 10: DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III.

FIG. 11: TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II.

FIG. 12: TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I.

FIG. 13: TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III.

FIG. 14: DVS forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II.

DETAILED DESCRIPTION

The following description sets forth exemplary compositions and methods,parameters and the like. It should be recognized, however, that suchdescription is not intended as a limitation on the scope of the presentdisclosure but is instead provided as a description of exemplaryembodiments.

JAK inhibitors CYT-0387,N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide,that is disclosed in U.S. Pat. No. 8,486,941 having the below structure:

The dihydrochloride anhydrous form of CYT-0387 is disclosed in PCTApplication WO 2012/071612. CYT-0387 dihydrochloride anhydrous has thebelow structure:

The XRPD, the differential scanning calorimetry (DSC), thethermogravimetric analysis (TGA), and the dynamic vapor sorption (DVS)of CYT-0387 dihydrochloride anhydrous form I (Form IV) are shown inFIGS. 1, 2, 3, and 4, respectively.

The present application provides other novel forms of CYT-387:monohydrochloride anhydrous Form I (Form I), dihydrochloride monohydrateForm II (Form II), and monohydrochloride anhydrous Form III (Form III).

As used herein, the terms “Form I” or “CYT-0387 Form I” are used torefer to CYT-0387 monohydrochloride anhydrous Form I; the terms “FormII” or “CYT-0387 Form II” are used to refer to CYT-0387 dihydrochloridehydrate, CYT-0387 dihydrochloride monohydrate, or CYT-0387dihydrochloride monohydrate Form II; and the terms “Form III” or“CYT-0387 Form III” are used to refer to CYT-0387 monohydrochlorideanhydrous Form III; and the terms “Form IV” or “CYT-0387 Form IV” areused to refer to CYT-0387 dihydrochloride anhydrous Form I.

In one embodiment, Form I or CYT-0387 Form I is characterized by XRPDshown in FIG. 6, DSC in FIG. 9, or TGA in FIG. 12. In one embodiment,Form I or CYT-0387 Form I has an XRPD pattern having peaks at about13.5°, 20.9°, 26.1°, 26.6°, and 28.3° 2-θ±0.2° 2-θ.

In some embodiment, Form II or CYT-0387 Form II is characterized by XRPDshown in FIG. 5, DSC in FIG. 8, TGA in FIG. 11, or DVS in FIG. 14. Inone embodiment, Form II or CYT-0387 Form II has an XRPD pattern havingpeaks at about 7.7°, 19.3°, 24.0°, 25.7°, and 29.6° 2-θ±0.2° 2-θ.

In other embodiment, Form III or CYT-0387 Form III is characterized byXRPD shown in FIG. 7, DSC in FIG. 10, or TGA in FIG. 13. In oneembodiment, Form III or CYT-0387 Form III has an XRPD pattern havingpeaks at about 12.7°, 14.6°, 17.8°, 19.7°, and 23.3° 2-θ±0.2° 2-θ.

In certain embodiment, Form IV or CYT-0387 Form IV is characterized byXRPD shown in FIG. 1, DSC in FIG. 2, or TGA in FIG. 3. In oneembodiment, Form IV or CYT-0387 Form IV has an XRPD pattern having peaksat about 5.5°, 10.1°, 14.9°, 25.1°, and 26.6° 2-θ±0.2° 2-θ.

Results of the present application found that CYT-0387 dihydrochloridemonohydrate (Form II) has the increased stability than other salts orforms of CYT-387 at certain conditions. The results described hereinalso found that such properties of CYT-387 Form II makes it moresuitable for developing or adopting to various synthesis or process. Inone embodiment, CYT-387 Form II is suitable for use in a pharmaceuticalcomposition in the tablet format. Also, the studies described hereinshowed the tablet formulation exhibited bioavailability propertiessimilar to those of capsule formulations. In certain embodiments, atablet comprising CYT-0387 dihydrochloride monohydrate Form II in anamount equivalent to 200 mg of free base of CYT-0387 provides similarbioavailability as a capsule comprising CYT-0387 dihydrochlorideanhydrous Form I in an amount equivalent to 300 mg of free base ofCYT-0387.

The results of present application indicate that CYT-0387dihydrochloride anhydrous Form I was hygroscopic and physically unstablewhen exposed to moisture. Also, the results described below indicatethat CYT-0387 dihydrochloride monohydrate Form II is a thermodynamicallystable form of the dihydrochloride salt under the conditions suitablefor manufacturing and/or storage. In addition, the present applicationdescribed the use of propyl gallate (a free radical scavenger oxidant)were effective in inhibiting or preventing oxidative degradation ofCYT-0387 dihydrochloride monohydrate Form II in an aqueous solution anda tablet formulation. Moreover, the results suggest that CYT-0387dihydrochloride monohydrate Form II exhibits increased bioavailabilitycompared to CYT-0387 dihydrochloride anhydrous Form I and CYT-0387 freebase.

In a still further embodiment, the present invention also providesamorphousN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride monohydrate. In an additional embodiment, thepresent invention also provides amorphousN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) monohydrochloride anhydrous and amorphousN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) monohydrochloride anhydrous. In another embodiment, thepresent invention also provides amorphousN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride and amorphousN-(cyanomethyl)-4-(2-(4-morpholinophenylamino) pyrimidin-4-yl)benzamide(CYT-0387) monohydrochloride.

In particular embodiments,N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride monohydrate Form II is in a crystalline form.

In further embodiments,N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) monohydrochloride anhydrous Form I is in a crystalline form.

In certain embodiments,N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) monohydrochloride anhydrous Form III is in a crystallineform.

In one embodiment, the crystalline forms are characterized by theinterlattice plane intervals determined by an X-ray powder diffractionpattern (XRPD). The diffractogram of XRPD is typically represented by adiagram plotting the intensity of the peaks versus the location of thepeaks, i.e., diffraction angle 2θ (two-theta) in degrees. Theintensities are often given in parenthesis with the followingabbreviations: very strong=vst; strong=st; medium=m; weak=w; and veryweak=vw. The characteristic peaks of a given XRPD can be selectedaccording to the peak locations and their relative intensity toconveniently distinguish this crystalline structure from others.

Those skilled in the art recognize that the measurements of the XRPDpeak locations and/or intensity for a given crystalline form of the samecompound will vary within a margin of error. The values of degree 2θallow appropriate error margins. Typically, the error margins arerepresented by “±”. For example, the degree 2θ of about “8.7±0.3”denotes a range from about 8.7+0.3, i.e., about 9.0, to about 8.7±0.3,i.e., about 8.4. Depending on the sample preparation techniques, thecalibration techniques applied to the instruments, human operationalvariation, and etc, those skilled in the art recognize that theappropriate error of margins for a XRPD can be ±0.5; ±0.4; ±0.3; ±0.2;±0.1; ±0.05; or less. In certain embodiments of the invention, the XRPDmargin of error is ±0.2.

Additional details of the methods and equipment used for the XRPDanalysis are described in the Examples section.

The XRPD peaks for CYT-0387 dihydrochloride anhydrous Form I, CYT-0387,dihydrochloride monohydrate Form II, CYT-0387 monohydrochlorideanhydrous Form I and CYT-0387 monohydrochloride anhydrous Form III canbe found below in Table 1.

TABLE 1 XRPD peaks for CYT-0387 forms CYT-0387 CYT-0387, CYT-0387CYT-0387 dihydrochloride dihydrochloride monohydrochloridemonohydrochloride anhydrous monohydrate anhydrous anhydrous Form I FormII Form I Form III Relative Relative Relative Relative PositionIntensity Position Intensity Position Intensity Position Intensity[°2Th.] [%] [°2Th.] [%] [°2Th.] [%] [°2Th.] [%] 5.5 31.0 7.7 33.7 13.515.3 12.7 85.0 10.1 100.0 19.3 43.7 20.9 100.0 14.6 50.0 14.9 66.5 24.0100.0 26.1 20.6 17.8 55.5 25.1 86.7 25.7 79.0 26.6 15.5 19.7 100.0 26.669.3 29.6 35.7 28.3 16.6 23.3 60.1

As used herein, the following words, phrases and symbols are generallyintended to have the meanings as set forth below, except to the extentthat the context in which they are used indicates otherwise.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”. In certain embodiments, the term “about” includes the indicatedamount±10%. In other embodiments, the term “about” includes theindicated amount±5%. In certain other embodiments, the term “about”includes the indicated amount±1%. Also, the singular forms “a” and “the”include plural references unless the context clearly dictates otherwise.Thus, e.g., reference to “the compound” includes a plurality of suchcompounds and reference to “the assay” includes reference to one or moreassays and equivalents thereof known to those skilled in the art.

“Pharmaceutically acceptable” or “physiologically acceptable” refer tocompounds, salts, compositions, dosage forms and other materials whichare useful in preparing a pharmaceutical composition that is suitablefor veterinary or human pharmaceutical use.

“Pharmaceutically acceptable salts” or “physiologically acceptablesalts” refer to salts of pharmaceutical compounds that retain thebiological effectiveness and properties of the underlying compound, andwhich are not biologically or otherwise undesirable. There are acidaddition salts and base addition salts. Pharmaceutically acceptable acidaddition salts may be prepared from inorganic and organic acids. Acidsand bases useful for reaction with an underlying compound to formpharmaceutically acceptable salts (acid addition or base addition saltsrespectively) are known to one of skill in the art. Similarly, methodsof preparing pharmaceutically acceptable salts from an underlyingcompound (upon disclosure) are known to one of skill in the art and aredisclosed in for example, Berge, at al. Journal of PharmaceuticalScience, January 1977 vol. 66, No. 1, and other sources. If thecompounds described herein are obtained as an acid addition salt, thefree base can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt,particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds.“Treatment” or “treating” is an approach for obtaining beneficial ordesired results including clinical results. Beneficial or desiredclinical results may include one or more of the following: a) inhibitingthe disease or condition (e.g., decreasing one or more symptomsresulting from the disease or condition, and/or diminishing the extentof the disease or condition); b) slowing or arresting the development ofone or more clinical symptoms associated with the disease or condition(e.g., stabilizing the disease or condition, preventing or delaying theworsening or progression of the disease or condition, and/or preventingor delaying the spread (e.g., metastasis) of the disease or condition);and/or c) relieving the disease, that is, causing the regression ofclinical symptoms (e.g., ameliorating the disease state, providingpartial or total remission of the disease or condition, enhancing effectof another medication, delaying the progression of the disease,increasing the quality of life, and/or prolonging survival.

The compounds described herein in which from 1 to n hydrogen atomsattached to a carbon atom may be replaced by a deuterium atom or D, inwhich n is the number of hydrogen atoms in the molecule. It is knownthat the deuterium atom is a non-radioactive isotope of the hydrogenatom. Such compounds may increase resistance to metabolism, and thus maybe useful for increasing the half-life of the compounds described hereinor pharmaceutically acceptable salts, isomers, prodrugs, or solvatesthereof, when administered to a mammal. See, e.g., Foster, “DeuteriumIsotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci.,5(12):524-527 (1984). Such compounds are synthesized by means well knownin the art, for example by employing starting materials in which one ormore hydrogen atoms have been replaced by deuterium. In someembodiments, the compounds described herein are CYT-0387 dihydrochloridemonohydrate Form II, CYT-0387 monohydrochloride anhydrous Form I,CYT-0387 monohydrochloride anhydrous Form III, CYT-0387 dihydrochlorideanhydrous Form IV, Compound 3, Compound 4, Compound 8, Compound 10,Compound 12, and Compound 13.

“Prevention” or “preventing” means any treatment of a disease orcondition that causes the clinical symptoms of the disease or conditionnot to develop. Compounds may, in some embodiments, be administered to asubject (including a human) who is at risk or has a family history ofthe disease or condition.

The terms “Subject” or “patient” refer to an animal, such as a mammal(including a human), that has been or will be the object of treatment,observation or experiment. The methods described herein may be useful inhuman therapy and/or veterinary applications. In some embodiments, thesubject is a mammal. In one embodiment, the subject is a human. “Humanin need thereof” refers to a human who may have or is suspect to havediseases or conditions that would benefit from certain treatment; forexample, being treated with the compounds according to the presentapplication. The terms “subject in need thereof” or “patient in needthereof” refer to a subject or a patient who may have, is diagnosed, oris suspected to have diseases, or disorders, or conditions that wouldbenefit from the treatment described herein. In certain embodiments, thesubject or patient who (i) has not received any treatment, (ii) hasreceived prior treatment and is not responsive or did not exhibitimprovement, or (iii) is relapse or resistance to prior treatment.

The term “therapeutically effective amount” of a compound of the presentapplication or a pharmaceutically acceptable salt, isomers, prodrug, orsolvate thereof, means an amount sufficient to effect treatment whenadministered to a subject, to provide a therapeutic benefit such asamelioration of symptoms or slowing of disease progression. Thetherapeutically effective amount may vary depending on the subject, anddisease or condition being treated, the weight and age of the subject,the severity of the disease or condition, and the manner ofadministering, which can readily be determined by one or ordinary skillin the art. In one embodiment, the therapeutic effective amount of thecompound described herein is 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg.

The term “inhibition” indicates a decrease in the baseline activity of abiological activity or process.

Pharmaceutical Compositions and Administration

Compounds provided herein are usually administered in the form ofpharmaceutical compositions. Thus, provides herein are alsopharmaceutical compositions that contain one or more of the compounds ofany of the formulae disclosed herein or a pharmaceutically acceptablesalt, isomers, prodrug, or solvate thereof, and one or morepharmaceutically acceptable vehicles selected from carriers, adjuvantsand excipients. Suitable pharmaceutically acceptable vehicles mayinclude, for example, inert solid diluents and fillers, diluents,including sterile aqueous solution and various organic solvents,permeation enhancers, solubilizers and adjuvants. Such compositions areprepared in a manner well known in the pharmaceutical art. See, e.g.,Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia,Pa. 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rdEd. (G. S. Banker & C. T. Rhodes, Eds.). As used herein, “solvate” isformed by the interaction of a solvent and a compound. Solvates of saltsof the compounds of any of the formulae described herein are alsoprovided. Hydrates of the compounds of any of the formulae are alsoprovided. Also, “prodrug” is defined in the pharmaceutical field as abiologically inactive derivative of a drug that upon administration tothe human body is converted to the biologically active parent drugaccording to some chemical or enzymatic pathway.

The pharmaceutical compositions may be administered in either single ormultiple doses. The pharmaceutical composition may be administered byvarious methods including, for example, rectal, buccal, intranasal andtransdermal routes. In certain embodiments, the pharmaceuticalcomposition may be administered by intra-arterial injection,intravenously, intraperitoneally, parenterally, intramuscularly,subcutaneously, orally, topically, or as an inhalant. In someembodiments, the pharmaceutical composition is administered orally.

One mode for administration is parenteral, for example, by injection.The forms in which the pharmaceutical compositions described herein maybe incorporated for administration by injection include, for example,aqueous or oil suspensions, or emulsions, with sesame oil, corn oil,cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose,or a sterile aqueous solution, and similar pharmaceutical vehicles.

Oral administration may be another route for administration of thecompounds described herein. Administration may be via, for example,capsule or enteric coated tablets. In making the pharmaceuticalcompositions that include at least one compound of any of the formulaedescribed herein or a pharmaceutically acceptable salt, prodrug, orsolvate thereof, the active ingredient is usually diluted by anexcipient and/or enclosed within such a carrier that can be in the formof a capsule, sachet, paper or other container. When the excipientserves as a diluent, it can be in the form of a solid, semi-solid, orliquid material, which acts as a vehicle, carrier or medium for theactive ingredient. Thus, the compositions can be in the form of tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, sterile injectablesolutions, and sterile packaged powders.

For preparing solid compositions such as tablets, the principal activeingredient may be mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of any of the above formulae or a pharmaceutically acceptablesalt, prodrug, or solvate thereof. When referring to thesepreformulation compositions as homogeneous, the active ingredient may bedispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

The tablets or pills of the compounds described herein may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action, or to protect from the acid conditions of the stomach.For example, the tablet or pill can include an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer thatserves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The specific dose level of a compound of the formulae described hereinfor any particular subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease in the subject undergoing therapy. Forexample, a dosage may be expressed as a number of milligrams of acompound of the formula per kilogram of the subject's body weight(mg/kg). Dosages of between about 0.01 and 200 mg/kg may be appropriate.In some embodiments, about 0.01 and 150 mg/kg may be appropriate. Inother embodiments a dosage of between 0.05 and 100 mg/kg may beappropriate. Normalizing according to the subject's body weight isparticularly useful when adjusting dosages between subjects of widelydisparate size, such as occurs when using the drug in both children andadult humans or when converting an effective dosage in a non-humansubject such as dog to a dosage suitable for a human subject.

The compounds of the present application or the compositions thereof maybe administered once, twice, three, or four times daily, using anysuitable mode described above. Also, administration or treatment withthe compounds according to any of the formulae described herein may becontinued for a number of days; for example, commonly treatment wouldcontinue for at least 7 days, 14 days, or 28 days, for one cycle oftreatment. Treatment cycles are generally known and are frequentlyalternated with resting periods of about 1 to 28 days, commonly about 7days or about 14 days, between cycles. The treatment cycles, in otherembodiments, may also be continuous.

In some embodiments, the forms or compositions thereof disclosed hereinis formulated for oral administration using pharmaceutically acceptablecarriers. Pharmaceutical compositions formulated for oral administrationcan be in the form of tablets, pills, capsules, cachets, dragees,lozenges, liquids, gels, syrups, slurries, elixirs, suspensions, orpowders.

Pharmaceutically Acceptable Carriers

The term “carrier” refers to diluents or fillers, disintegrants,precipitation inhibitors, surfactants, glidants, binders, lubricants,anti-oxidants, and other excipients and vehicles with which the compoundis administered. Carriers are generally described herein and also in“Remington's Pharmaceutical Sciences” by E. W. Martin. Examples ofcarriers include, but are not limited to, aluminum monostearate,aluminum stearate, carboxymethylcellulose, carboxymethylcellulosesodium, crospovidone, glyceryl isostearate, glyceryl monostearate,hydroxyethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxyoctacosanyl hydroxystearate, hydroxypropyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, lactosemonohydrate, magnesium stearate, mannitol, microcrystalline cellulose,poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 188, poloxamer237, poloxamer 407, povidone, silicon dioxide, colloidal silicondioxide, silicone, silicone adhesive 4102, and silicone emulsion. Itshould be understood, however, that the carriers selected for thepharmaceutical compositions provided in the present disclosure, and theamounts of such carriers in the composition, may vary depending on themethod of formulation (e.g., dry granulation formulation, soliddispersion formulation).

The term “diluent” or “filler” generally refers to a substance that isused to dilute the compound of interest prior to delivery. Diluents canalso serve to stabilize compounds. Examples of diluents may includestarch, saccharides, disaccharides, sucrose, lactose, polysaccharides,cellulose, cellulose ethers, hydroxypropyl cellulose, sugar alcohols,xylitol, sorbitol, maltitol, microcrystalline cellulose, calcium orsodium carbonate, lactose, lactose monohydrate, dicalcium phosphate,cellulose, compressible sugars, dibasic calcium phosphate dehydrate,mannitol, microcrystalline cellulose, and tribasic calcium phosphate.

The term “disintegrant” generally refers to a substance which, uponaddition to a solid preparation, facilitates its break-up ordisintegration after administration and permits the release of an activeingredient as efficiently as possible to allow for its rapiddissolution. Examples of disintegrants may include maize starch, sodiumstarch glycolate, croscarmellose sodium, crospovidone, microcrystallinecellulose, modified corn starch, sodium carboxymethyl starch, povidone,pregelatinized starch, and alginic acid.

The term “precipitation inhibitors” generally refers to a substance thatprevents or inhibits precipitation of the active agent. One example of aprecipitation inhibitor includes hydroxypropylmethylcellulose.

The term “surfactants” generally refers to compounds that lower thesurface tension between two liquids or between a liquid and a solid.Examples of surfactants include poloxamer and sodium lauryl sulfate.

The term “glidant” generally refers to substances used in tablet andcapsule formulations to improve flow-properties during tabletcompression and to produce an anti-caking effect. Examples of glidantsmay include colloidal silicon dioxide, talc, fumed silica, starch,starch derivatives, and bentonite.

The term “binder” generally refers to any pharmaceutically acceptablefilm which can be used to bind together the active and inert componentsof the carrier together to maintain cohesive and discrete portions.Examples of binders may include hydroxypropylcellulose,hydroxypropylmethylcellulose, povidone, copovidone, ethyl cellulose,gelatin, and polyethylene glycol.

The term “lubricant” generally refers to a substance that is added to apowder blend to prevent the compacted powder mass from sticking to theequipment during the tableting or encapsulation process. A lubricant canaid the ejection of the tablet form the dies, and can improve powderflow. Examples of lubricants may include magnesium stearate, stearicacid, silica, fats, calcium stearate, polyethylene glycol, sodiumstearyl fumarate, or talc; and solubilizers such as fatty acidsincluding lauric acid, oleic acid, and C₈/C₁₀ fatty acid.

The term “anti-oxidant” generally refers to a substance that inhibitsthe oxidation of other substances. In certain embodiments of theinvention, anti-oxidants are added to the pharmaceutical composition.Examples of anti-oxidants may include ethylenediaminetetraacetic acid,ethylenediaminetetraacetic acid disodium salt, sodium sulfite, sodiummetabisulfite, sodium bisulfite, butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), ascorbic acid, ascorbyl palmitate,thioglycerol, thioglycolic acid, tocopherol (vitamin E), D-α tocopherylpolyethylene glycol 1000 succinate (vitamin E TPGS) and propyl gallate.In certain embodiment, the antioxidant is propyl gallate. In oneembodiment, the pharmaceutical composition comprises CYT-0387dihydrochloride monohydrate Form II and an antioxidant selected from thegroup consisting of butylated hydroxyanisole (BHA), ascorbic acid, andpropyl gallate. In certain embodiment, the pharmaceutical compositioncomprises CYT-0387 dihydrochloride monohydrate Form II and anantioxidant, wherein the antioxidant is propyl gallate.

The anti-oxidant or antioxidant may be present in an amount that issufficient to prevent, inhibit, and/or reduce degradation of the activeingradient (such as CYT-0387 Form II). By way of examples, theantioxidant may be present in an amount of about 0.001%, about 0.002%,about 0.005%, about 0.01%, about 0.02%, about 0.05%, about 0.1%, about0.2%, about 0.5%, or about 1%. In one embodiment, the pharmaceuticalcomposition comprises propyl gallate at an amount of about 0.001%, about0.01%, about 0.1%, about 0.2%, about 0.5%, or about 1%. In someembodiment, the pharmaceutical composition comprises CYT-0387dihydrochloride monohydrate Form II and about 0.2% of propyl gallate.

In certain aspects, provided is a pharmaceutical composition comprisingat least one active agent (including, for example, CYT-0387dihydrochloride monohydrate Form II), and one or more of (a)-(e): a) atleast one diluent; b) at least one disintegrant; c) at least oneglidant; d) at least one lubricant; and e) at least one anti-oxidant.

In some embodiments, the pharmaceutical composition comprises at leastone or at least two diluent(s). In certain embodiments, thepharmaceutical composition comprises one or two diluent(s). In certainembodiments, the diluent is selected from the group consisting ofmannitol, microcrystalline cellulose, lactose, dextrose, sucrose,ludiflash, F-melt, advantose, GalenIQ, or any mixtures thereof. In oneembodiment, the diluent is mannitol, microcrystalline cellulose, or amixture thereof.

In some embodiments, the pharmaceutical composition comprises at leastone disintegrant. In certain embodiments, the pharmaceutical compositioncomprises one disintegrant. In a particular embodiment, the disintegrantis sodium starch glycolate. In one embodiment, the disintegrant iscroscarmellose sodium. In another embodiment, the disintegrant iscrospovidone.

In some embodiments, the pharmaceutical composition comprises at leastone glidant. In certain embodiments, the pharmaceutical compositioncomprises one glidant. In one embodiment, the glidant is colloidalsilicon dioxide.

In some embodiments, the pharmaceutical composition comprises at leastone lubricant. In certain embodiments, the pharmaceutical compositioncomprises one lubricant. In one embodiment, the lubricant is magnesiumstereate.

In particular embodiments, the pharmaceutical composition comprisesCYT-0387 dihydrochloride monohydrate Form II, at least one diluent, atleast one disintegrant, at least one glidant, at least one lubricant,and at least one anti-oxidant. In further embodiments, the at least onediluent is microcrystalline cellulose, the at least one disintegrant issodium starch glycolate, the at least one glidant is colloidal silicondioxide, the at least one lubricant is magnesium stearate, and at leastone anti-oxidant is propyl gallate. In yet further embodiments, the atleast one diluent is lactose, the at least one disintegrant is sodiumstarch glycolate, the at least one glidant is colloidal silicon dioxide,the at least one lubricant is magnesium stearate, and at least oneanti-oxidant is propyl gallate.

In other embodiments, the pharmaceutical composition comprises CYT-0387dihydrochloride monohydrate Form II, at least two diluents, at least onedisintegrant, at least one glidant, at least one lubricant, and at leastone anti-oxidant. In yet other embodiments, the at least two diluentsare microcrystalline cellulose and lactose, the at least onedisintegrant is sodium starch glycolate, the at least one glidant iscolloidal silicon dioxide, the at least one lubricant is magnesiumstearate, and at least one anti-oxidant is propyl gallate.

In certain embodiments, the pharmaceutical composition comprisesCYT-0387 of which at least about 80% is CYT-0387 dihydrochloridemonohydrate Form II. In further embodiments, the pharmaceuticalcomposition comprises CYT-0387 of which at least about 85% is CYT-0387dihydrochloride monohydrate Form II. In still further embodiments, thepharmaceutical composition comprises CYT-0387 of which at least about90% is CYT-0387 dihydrochloride monohydrate Form II. In yet furtherembodiments, the pharmaceutical composition comprises CYT-0387 of whichat least about 95% is CYT-0387 dihydrochloride monohydrate Form II. Inparticular embodiments, the pharmaceutical composition comprisesCYT-0387 of which at least about 97% is CYT-0387 dihydrochloridemonohydrate Form II. In other embodiments, the pharmaceuticalcomposition comprises CYT-0387 of which at least about 98% is CYT-0387dihydrochloride monohydrate Form II. In still other embodiments, thepharmaceutical composition comprises CYT-0387 of which at least about99% is CYT-0387 dihydrochloride monohydrate Form II. In yet otherembodiments, the pharmaceutical composition comprises CYT-0387 of whichat least about 99.5% is CYT-0387 dihydrochloride monohydrate Form II. Inparticular embodiments, the pharmaceutical composition comprisesCYT-0387 of which at least about 99.9% is CYT-0387 dihydrochloridemonohydrate Form II.

It should be understood that the pharmaceutical composition comprisespharmaceutically acceptable carriers detailed herein, the same as ifeach and every combination of pharmaceutically acceptable carrier werespecifically and individually listed.

Unit Dosage Forms

In some embodiments, the pharmaceutical compositions as described hereinare formulated in a unit dosage form. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forsubjects (e.g., human subjects and other mammals), each unit containinga predetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical carrier.

In a further embodiment, the invention is directed to unit dosage formscomprising CYT-0387 dihydrochloride monohydrate Form II. In someembodiments, the unit dosage form comprises CYT-0387 dihydrochloridemonohydrate Form II in amount equivalent to from about 10 mg to about1000 mg, about 10 mg to about 800 mg, about 10 mg to about 700 mg about10 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about10 mg to about 150 mg, about 10 mg to about 100 mg, about 10 mg to about50 mg, about 50 mg to about 1000 mg, about 50 mg to about 800 mg, about50 mg to about 700 mg about 50 mg to about 500 mg, about 50 mg to about400 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about100 mg, about 100 mg to about 1000 mgs, about 100 mg to about 800 mg,about 100 mg to about 700 mg about 100 mg to about 500 mg, about 100 mgto about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250mg, about 100 mg to about 200 mg, about 150 mg to about 300 mg, about150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg toabout 300 mg, about 200 mg to about 250 mg, or about 200 mg to about 300mg of CYT-0387 free base.

In additional embodiments, the dosage form comprises CYT-0387dihydrochloride monohydrate Form II in an amount equivalent to about 100mg of CYT-0387 free base, about 150 mg CYT-0387 free base, 200 mg ofCYT-0387 free base, about 250 mg CYT-0387 free base, 300 mg of CYT-0387free base, about 400 mg CYT-0387 free base, or about 500 mg of CYT-0387free base. In certain embodiments, the unit dosage form comprisesCYT-0387 dihydrochloride monohydrate Form II in an amount equivalent toabout 50 mg of CYT-0387 free base. In other embodiments, the unit dosageform comprises CYT-0387 dihydrochloride monohydrate Form II in an amountequivalent to about 100 mg of CYT-0387 free base. In yet otherembodiments, the unit dosage form comprises CYT-0387 dihydrochloridemonohydrate Form II in an amount equivalent to about 150 mg of CYT-0387free base. In still other embodiments, the dosage form comprisesCYT-0387 dihydrochloride monohydrate Form II in an amount equivalent toabout 200 mg of CYT-0387 free base. In particular embodiments, thedosage form comprises CYT-0387 dihydrochloride monohydrate Form II in anamount equivalent to about 250 mg of CYT-0387 free base. In furtherembodiments, the dosage form comprises CYT-0387 dihydrochloridemonohydrate Form II in an amount equivalent to about 300 mg of CYT-0387free base. In yet further embodiments, the dosage form comprisesCYT-0387 dihydrochloride monohydrate Form II in an amount equivalent toabout 400 mg of CYT-0387 free base. In still further embodiments, thedosage form comprises CYT-0387 dihydrochloride monohydrate Form II in anamount equivalent to about 500 mg of CYT-0387 free base. In otherembodiments, the pharmaceutical composition is a tablet at a dosage formcomprising CYT-0387 dihydrochloride monohydrate Form II in an amountequivalent to about 300 mg of CYT-0387 free base.

In further embodiments, the invention is directed to dosage formscomprising CYT-0387 dihydrochloride monohydrate Form II in an amountequivalent to 200 mg of the free baseN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidewhich provide a pharmacokinetic profile substantially similar to adosage form comprising CYT-0387 dihydrochloride anhydrous Form I in anamount equivalent to 300 mg of the free baseN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide.

In certain embodiments of the invention, the unit dosage form comprisesat least one pharmaceutically acceptable carrier. In other embodiments,the unit dosage form comprises CYT-0387 dihydrochloride monohydrate FormII, at least two diluents, at least one disintegrant, at least oneglidant, at least one lubricant, and at least one anti-oxidant. In stillfurther embodiments, the unit dosage form comprises about 36% to 44%CYT-0387 dihydrochloride monohydrate Form II; about 44% to 58% diluent;about 4% to 8% disintegrant, about 0.25% to 0.75% glidant, about 1.2% to1.8% lubricant, and about 0.1% to 0.5% anti-oxidant. In yet otherembodiments, the at least two diluents are microcrystalline celluloseand lactose, the at least one disintegrant is sodium starch glycolate,the at least one glidant is colloidal silicon dioxide, the at least onelubricant is magnesium stearate, and at least one anti-oxidant is propylgallate. In still further embodiments, the unit dosage form comprisesabout 36% to 44% CYT-0387 dihydrochloride monohydrate Form II; about 30%to 38% microcrystalline cellulose; about 14% to 20% lactose, about 4% to8% sodium starch glycolate, about 0.25% to 0.75% colloidal silicondioxide, about 1.2% to 1.8% magnesium stearate, and about 0.1% to 0.5%propyl gallate.

Manufacturing of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be manufacturedusing any conventional method, such as, but not limited to, mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, melt-spinning, spray-drying, or lyophilizingprocesses.

A skilled artisan would recognize suitable methods and techniques toprepare a tablet by conventional formulation. Exemplary methods andtechniques to prepare powders for compression into a tablet include drygranulation or wet granulation. Dry granulation generally refers to theprocess of forming granules without using a liquid solution, whereas wetgranulation generally refers to the process of adding a liquid solutionto powders to granulate.

Kits

Provided herein are also kits that include a compound of the formulae ofthe present application or a pharmaceutically acceptable salt, isomer,prodrug, or solvate thereof, and suitable packaging. In one embodiment,a kit further includes instructions for use. In one aspect, a kitincludes a compound of the formulae described herein or apharmaceutically acceptable salt, isomer, prodrug, or solvate thereof,and a label and/or instructions for use of the compounds in thetreatment of the indications, including the diseases or conditions,described herein.

Provided herein are also articles of manufacture that include a compoundof any of the formulae described herein or a pharmaceutically acceptablesalt, isomer, prodrug, or solvate thereof, in a suitable container. Thecontainer may be a vial, jar, ampoule, preloaded syringe, andintravenous bag.

Methods of Treatment

The CYT-0387 forms of the present invention may be used in the treatmentof kinase associated diseases including JAK kinase associated diseasessuch as immunological and inflammatory diseases including organtransplants; hyperproliferative diseases including cancer andmyeloproliferative diseases; viral diseases; metabolic diseases; andvascular diseases.

In addition to primates, such as humans, a variety of other mammals canbe treated using the compounds, compositions and methods of the presentinvention. For instance, mammals including, but not limited to, cows,sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine,ovine, equine, canine, feline, rodent or murine species can be treated.However, the invention can also be practiced in other species, such asavian species (e.g., chickens).

The term “administering” should be understood to mean providing acompound of the invention to a subject in need of treatment.

The terms “treating” or “treatment” refer to obtaining a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. Treatment may cover anytreatment of a disease in a mammal, and includes: preventing the diseasefrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it; inhibiting the disease, i.e.,arresting its development; or relieving the disease, i.e., causingregression of the disease. The therapeutic agent may be administeredbefore, during or after the onset of disease. The treatment of ongoingdisease, where the treatment stabilizes or reduces the undesirableclinical symptoms of the patient, is of particular interest. Theexpected progression-free survival times can be measured in months toyears, depending on prognostic factors including the number of relapses,stage of disease, and other factors. Prolonging survival includeswithout limitation times of at least 1 month, about at least 2 months,about at least 3 months, about at least 4 months, about at least 6months, about at least 1 year, about at least 2 years, about at least 3years, or more. Overall survival can also be measured in months toyears. The patient's symptoms may remain static or may decrease.

The term “effective amount” refers to an amount that may be effective toelicit the desired biological or medical response, including the amountof a compound that, when administered to a subject for treating adisease, is sufficient to effect such treatment for the disease. Theeffective amount will vary depending on the compound, the disease andits severity and the age, weight, etc., of the subject to be treated.The effective amount can include a range of amounts.

The term “kinase associated diseases” refers to a disorder or disordersthat directly or indirectly result from or are aggravated by aberrantkinase activity, in particular JAK activity and/or which are alleviatedby inhibition of one or more of these kinase enzymes.

In a preferred embodiment the kinase associated disease state involvesone or more of the JAK kinases, JAK1, JAK2, JAK3 or TYK2. In aparticularly preferred embodiment, the disease involves JAK2 kinase.Such diseases include, but are not limited to, those listed in the Tablebelow.

Activation of the JAK/STAT Pathway in Various Pathologies Cell TypesCytokines JAK Kinase Disease Type Involved involved InvolvedCharacteristics Atopy Allergic Asthma, Mast Cells, IL-4, IL-5, IL-6,JAK1, T-cell activation of Atopic Dermatitis Eosinophils, IL-7, IL-13JAK2, B-cells followed by (Eczema), T-Cells, B-Cells, JAK3, IgE mediatedAllergic Rhinitis, Tyk2 activation of resident Mast cells andEosinophils CMI Allergic Contact T-cells, B-cells, IL-2, IL-4, IL-5,JAK1, B cell and/or T_(DH) Dermatitis, macrophages, IL-6, IL-10, JAK2,cell activation hypersensitivity Neutrophils IFNγ, TNF, JAK3,Macrophage/granulocyte pneumonitis IL-7, IL-13, Tyk2 activationAutoImmune Diseases Multiple sclerosis, B-Cells, T cells, IL-2, IL-4,IL-5, JAK1, Cytokine Production Glomerulonephritis monocytes, IL-6,IL-7, JAK2, (e.g. TNFα/β, IL-1, Systemic Lupus Macrophages, Il-10,IL-13, JAK3, CSF-1, GM-CSF), T-cell Erythematosus Neutrophils, IFNγ,TNF, Tyk2 Activation, B cell (SLE), Rheumatoid Mast Cells, GM-CSF;G-CSF, activation, Arthritis, Juvenile Eosinophils, JAK/STAT activationArthritis, Sjögren's Syndrome, Scleroderma Polymyositis, AnkylosingSpondylitis, Psoriatic Arthritis Transplantation Allograft Rejection Tcells, B cells, IL-2, IL-4, IL-5, JAK1, Macrophage/T cell GvHDMacrophages IL-7, IL-13, JAK2, mediated necrosis, TNF JAK3, Tc cellmediated apoptosis, and B cell/Ig mediated opsonization/necrosis offoreign graft Viral Diseases Epstein Barr Virus Lymphocytes Viral JAK1,JAK/STAT (EBV) Cytokines, IL-2, JAK2, Mediation Hepatitis B HepatocytesJAK3 Hepatitis C Hepatocytes HIV Lymphocytes HTLV 1 LymphocytesVaricella-Zoster Fibroblasts Virus (VZV) Human Papilloma Epithelialcells Virus (HPV) Hyperproliferative diseases-cancer Leukemia LeucocytesVarious JAK1, Cytokine production, Lymphoma Lymphocytes Autocrine JAK2,JAK/STAT Multiple Myeloma Various cytokines, JAK3 Activation prostatecancer Various Intrinsic breast cancer Various Activation hodgkinslympohoma Various B-cell chronic Various lymphocytic leukemia lungcancer Various hepatoma Various metastatic melanoma Various gliomaVarious Myeloproliferative Diseases Polycythemia vera HematopoieticInterleukin-3, JAK2 JAK/STAT activation (PV), primary erythropoietin,mutation myelofibrosis, thrombopoietin thrombocythemia, essentialthrombocythemia (ET), idiopathic myelofibrosis, chronic myelogenousleukemia, systemic mastocystosis (SM), chronic neutrophilic leukemia(CNL), myelodisplastic syndrome (MDS), systemic mast cell disease (SMCD)Vascular Disease Hypertension, Endothelial cells, IL6, JAK1, JAK/STATactivation Hypertrophy, Heart smooth muscle angiotensin II, JAK2,Failure, Ischemia, cells including LIF, TNFalpha, TYK2 Pulmonaryarterial pulmonary artery serotonin, hypertension smooth musclecaveolin1 cells, cardiac myocytes, fibroblasts, endothelial cellsMetabolic disease Obesity, metabolic Adipocytes, Leptin JAK2 JAK/STATactivation syndrome pituitary cells, neurons, Monocytes

The term “immunological and inflammatory disease” refers to animmunological, inflammatory or autoimmune disease, including but notlimited to rheumatoid arthritis, polyarthritis, rheumatoid spondylitis,osteoarthritis, gout, asthma, bronchitis, allergic rhinitis, chronicobstructive pulmonary disease, cystic fibrosis, inflammatory bowldisease, irritable bowl syndrome, mucous colitis, ulcerative colitis,diabrotic colitis, Crohn's disease, autoimmune thyroid disorders,gastritis, esophagitis, hepatitis, pancreatitis, nephritis, psoriasis,eczema, acne vulgaris, dermatitis, hives, multiple sclerosis,Alzheimer's disease, Motor Neurone Disease (Lou Gehrig's disease),Paget's disease, sepsis, conjunctivitis, neranl catarrh, chronicarthrorheumatism, systemic inflammatory response syndrome (SIRS),polymyositis, dermatomyositis (DM), Polaritis nodoa (PN), mixedconnective tissue disorder (MCTD), Sjoegren's syndrome, Crouzonsyndrome, achondroplasia, systemic lupus erythematosus, scleroderma,vasculitis, thanatophoric dysplasia, insulin resistance, Type I diabetesand complications from diabetes and metabolic syndrome.

The term “hyperproliferative diseases” includes cancer andmyeloproliferative disease states such as cellular-proliferative diseasestates, including but not limited to: Cardiac: sarcoma (angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamouscell, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hanlartoma, inesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostrate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfrorna (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformians), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, SertoliLeydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma]),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma; Skin: malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, psoriasis; Adrenal glands: neuroblastoma; andMyeloproliferative diseases such as polycythemia vera (PV), primarymyelofibrosis, thrombocythemia, essential thrombocythemia (ET), agnoneicmyeloid metaplasia (AMM), also referred to as idiopathic myelofibrosis(IMF), chronic myelogenous leukemia (CML), systemic mastocystosis (SM),chronic neutrophilic leukemia (CNL), myelodisplastic syndrome (MDS) andsystemic mast cell disease (SMCD). In certain embodiment, themyelofibrosis disease is selected from polycythemia vera (PV), primarymyelofibrosis, thrombocythemia, and essential thrombocythemia (ET). Inone embodiment, the pharmaceutical composition of the presentapplication may be suitable for treating myeloproliferative diseases,wherein the myelofibrosis disease is selected from polycythemia vera(PV), primary myelofibrosis, thrombocythemia, and essentialthrombocythemia (ET). In some embodiment, the pharmaceutical compositionof the present application may be suitable for treating primarymyelofibrosis.

The term “vascular diseases” refers to diseases including but notlimited to cardiovascular diseases, hypertension, hypertrophy,hypercholesterolemia, hyperlipidemia, thrombotic disorders, stroke,Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine,peripheral arterial disease, heart failure, restenosis, atherosclerosis,left ventricular hypertrophy, myocardial infarction, ischemic diseasesof heart, kidney, liver and brain, and pulmonary arterial hypertension.

Preferred diseases for JAK2 selective inhibitors include immunologicaland inflammatory diseases such as auto-immune diseases for exampleatopic dermatitis, asthma, rheumatoid arthritis, Crohn's disease,psoriasis, Crouzon syndrome, achondroplasia, systemic lupuserythematosus, scleroderma, mixed connective tissue disease, vasculitis,thanatophoric dysplasia and diabetes; hyperproliferative disorders suchas cancer for example prostate cancer, colon cancer, breast cancer,liver cancer such as hepatoma, lung cancer, head and neck cancer such asglioma, skin cancer such as metastatic melanoma, leukemia, lymphoma,multiple myeloma and myeloproliferative diseases such as polycythemiavera (PV), myelofibrosis, thrombocythemia, essential thrombocythemia(ET), agnoneic myeloid metaplasia (AMM), also referred to as idiopathicmyelofibrosis (IMF) and chronic myelogenous leukemia (CML); and vasculardiseases such as hypertension, hypertrophy, stroke, Raynaud'sphenomenon, POEMS syndrome, angina, ischemia, migraine, peripheralarterial disease, heart failure, restenosis, atherosclerosis andpulmonary arterial hypertension.

In other embodiments, the disease is a solid tumor. By way of examples,the solid tumor includes but is not limited to pancreatic cancer,bladder cancer, colorectal cancer, breast cancer, prostate cancer, renalcancer, hepatocellular cancer, lung cancer, ovarian cancer, cervicalcancer, rectum cancer, liver cancer, kidney cancer, stomach cancer, skincancer, gastric cancer, esophageal cancer, head and neck cancer,melanoma, neuroendocrine cancers, CNS cancers (e.g., neuroblastoma),brain tumors (e.g., glioma, anaplastic oligodendroglioma, adultglioblastoma multiforme, and adult anaplastic astrocytoma), bone cancer,or soft tissue sarcoma. In some embodiments, the solid tumor isnon-small cell lung cancer, small-cell lung cancer, colon cancer, CNScancer, melanoma, ovarian cancer, renal cancer, pancreatic cancer,prostate cancer, or breast cancer. In particular embodiments, the solidtumor is non-small cell lung cancer, colon cancer, pancreatic cancer, orbreast cancer. In further embodiments, the solid tumor is non-small celllung cancer. In still further embodiments, the solid tumor is coloncancer. In yet further embodiments, the solid tumor is pancreaticcancer. In even further embodiments, the solid tumor is breast cancer.

In further methods of the present invention, CYT-0387, or a formthereof, including CYT-0387 dihydrochloride monohydrate Form II, is usedto maintain or elevate hemoglobin levels in a subject experiencinganemia or a hemoglobin decline. Anemic subjects have an endogenoushemoglobin level that is lower than the level that is normal for healthysubject of equivalent age and gender. Acceptable or “normal” levels arenow well established in medical practice. For an adult human male,anemia is evident when the hemoglobin level is below about 13.0 g/dL;for non-pregnant adult human females, deficiency is evident when thehemoglobin level is below about 12.0 g/dL. Measurement of hemoglobinlevels is performed using well established techniques. Conditions ofsevere anemia are evident when the hemoglobin level is less than about8.0 g/dL.

In use, CYT-0387 is administered to an anemic subject in an amounteffective to maintain or elevate the level of hemoglobin in the subject.Administration of the drug thus has the minimum effect of inhibitingfurther reduction in the level of hemoglobin in the treated subject.More desirably, administration of the drug has the effect of increasingthe level of hemoglobin in the subject.

Anemic subjects that would benefit from treatment with CYT-0387 includesubjects that have undergone or are undergoing chemotherapy or radiationtherapy, such as cancer patients. A wide variety of chemotherapeuticagents are known to have the consequence of reducing the level offunctioning red blood cells. As well, subjects that are CYT-0387treatment candidates are those afflicted with blood disorders includingblood cancers that result in, or are associated with, a reduction in redblood cell count. In embodiments, the subjects to be treated aresubjects having anemia associated with or resulting from such bloodconditions as myelodysplastic syndrome. Myelodysplasia syndromes (MDS)is a term used to describe a group of diseases characterized byineffective hematopoiesis leading to blood cytopenias and hypercellularbone marrow. MDS has traditionally been considered to be synonymous with‘preleukemia’ because of the increased risk of transformation into acutemyelogenous leukemia (AML). Evolution to AML and the clinicalconsequences of cytopenias are main causes of morbidity and mortality inMDS. Debilitating symptoms of MDS include fatigue, pallor, infection,and bleeding. Anemia, neutropenia, and thrombocytopenia are also commonclinical manifestations of MDS. In other embodiments, the subjects to betreated are subjects having anemia associated with or resulting fromsuch other blood conditions as anemias associated with other hematologicmalignancies, aplastic anemia, anemia of chronic disease that affect redblood cells and the like. Anemia of chronic disease is associated withsuch diseases as certain cancers including lymphomas and Hodgkin'sdisease; autoimmune diseases such as rheumatoid arthritis, systemiclupus erythematosis, inflammatory bowel disease and polymyalgiarheumatica; long term infections such as urinary tract infection, HIVand osteomyelitis; heart failure; and chronic kidney disease. Inaddition, patients with anemia resulting from conditions associated withincreased destruction, shortened red blood cell survival and splenicsequestration could also benefit from CYT-0387 treatment. Patientsafflicted with these conditions thus can be treated to improve upontheir state of declining or deficient hemoglobin.

In certain embodiments, the subject to be treated is an anemic subjectexperiencing thalassemia. In other embodiments, the subject to betreated is a subject other than a subject experiencing thalassemia.

In embodiments, a CYT-0387 form of the present invention, such asCYT-0387 dihydrochloride monohydrate Form II, is administered to asubject diagnosed with a myeloproliferative disease such asmyeloproliferative neoplasm, thereby to improve upon the prognosis ofthe disease and, in embodiments, particularly to treat hemoglobindeficiency or decline associated with the disease. In other embodiments,a CYT-0387 form of the present invention, such as CYT-0387dihydrochloride monohydrate Form II, is administered to an anemicsubject that is other than an anemic subject diagnosed with amyeloproliferative disease. This class of treatable subject presentswith anemia unrelated to myeloproliferative disease. In someembodiments, a CYT-0387 form of the present application, such asCYT-0387 dihydrochloride monohydrate Form II, is administered to ananemic subject that is diagnosed with cancer.

“Myeloproliferative diseases” and “myeloproliferative neoplasms (MPN)”most notably polycythemia vera (PV), essential thrombocythemia (ET) andprimary myelofibrosis (PMF) are a diverse but inter-related group ofclonal disorders of pluripotent hematopoietic stem cells that share arange of biological, pathological and clinical features including therelative overproduction of one or more cells of myeloid origin, growthfactor independent colony formation in vitro, marrow hypercellularity,extramedullary hematopoiesis, spleno- and hepatomegaly, and thromboticand/or hemorrhagic diathesis. An international working group formyeloproliferative neoplasms research and treatment (IWG-MRT) has beenestablished to delineate and define these conditions (see for instanceVannucchi et al, CA Cancer J. Clin., 2009, 59: 171-191), and thosedisease definitions are to be applied for purposes of thisspecification. Subjects, most notably human patients, who present withMPN and particularly PMF are identifiable in the art using the IWG-MRTcriteria mentioned above. Subjects “at risk for” a particular form ofMPN are subjects having an early stage form of the disease, and may forinstance include subjects having a genetic marker thereof, such as theJAK2V617F allele which is associated with PV (>95%), with ET (60%) andwith PMF (60%). Subjects are also considered to be “at risk for” a formof MFN if they already manifest symptoms of an earlier stage form. Thus,subjects presenting with MFN are at risk for post-PV and post-ET, bothof which develop following MPN.

The response of MPN patients and particularly PMF patients to CYT-0387therapy is particularly robust when, according to the present invention,they are patients selected for CYT-0387 therapy based on one or more ofthe following criteria:

-   -   i. prior therapy with a drug selected from thalidomide,        lenalidomide, pomalidomide and a JAK2 inhibitor other than        CYT-0387;    -   ii. a clinical criterion selected from one or both of (1)        smaller spleen size and (2) a lower percentage of circulating        blasts;    -   iii. a biochemical marker criterion selected from one or more        of (1) an increased level of at least one protein selected from        EGF, TNF-α, G-CSF, IFN-α, MIP-133, HGF, MIG, and VEGF; (2) a        decreased level of eotaxin; and (3) an altered level of at least        one protein selected from EPO, hepcidin and BMP-2.

The improved outcome from CYT-0387 therapy that results from priorpatient selection is manifested as a robust improvement in anemiaresponse and/or in spleen response. By “anemia response” is meant anincrease in the patient's hemoglobin level or a patient who wastransfusion dependent becoming transfusion independent. Desirably, aminimum increase in hemoglobin of 2.0 g/dL lasting a minimum of 8 weeksis achieved, which is the level of improvement specified in theInternational Working Group (IWG) consensus criteria. However, smaller,but still medically significant, increases in hemoglobin are alsoconsidered to be within the term “anemia response”.

By “spleen response” is meant a reduction in the size of the patient'sspleen as assessed by either palpation of a previously palpable spleenduring physical exam or by diagnostic imaging. The IWG consensuscriteria specifies that there be either a minimum 50% reduction inpalpable splenomegaly (spleen enlargement) of a spleen that is at least10 cm at baseline (prior to treatment) or of a spleen that is palpableat more than 5 cm at baseline becomes not palpable. However, smallerreductions are also considered to be within the term “spleen response”.

In one embodiment, the selected patient is one that has received priordrug therapy. More particularly, patients selected for CYT-0387 therapyinclude patients that have been treated, or are currently being treated,with thalidomide (CAS number 50-35-1) or with a derivative thereof,particularly lenalidomide (CAS number 191732-72-6). These drugs are bothused in the treatment of multiple myeloma, and appear also to be showingsome benefit in patients afflicted with myeloproliferative disorder. Toreceive the further benefit resulting from subsequent CYT-0387 therapy,patients will either be undergoing treatment with thalidomide,lenalidomide or pomalidomide or similar agent or will have been treatedwith one of these drugs within a time frame, relative to CYT-0387therapy onset, sufficient for the effects of these drugs to be manifest.Patients meeting these criteria experience significant anemia response,relative to patients naive to this drug therapy, when subsequentlytreated with CYT-0387. In a preferred embodiment, the CYT-0387 patientis one subjected to prior therapy with lenalidomide.

Patients selected for CYT-0387 therapy also include patients that havebeen treated, or are undergoing treatment, with a JAK inhibitor otherthan CYT-0387. It has been found in particular that patients previouslytreated with the JAK inhibitor designated INCBO 18424, or the JAKinhibitor designated TG101348, have a more prominent spleen response toCYT-0387 therapy than patients naive to such prior therapy. In apreferred embodiment, the patient selected for CYT-0387 therapy is onethat, in addition to being subjected to therapy with a JAK inhibitorother than CYT-0387, is also a transfusion dependent patient. INCBO18424 is administered at starting doses of 15 or 20 mg po BID with dosetitration from 5 mg BID to 25 mg BID. TG101348 is administered once aday with a maximum tolerated dose (MTD) determined to be 680 mg/day. JAKinhibitors other than CYT-0387 include all and any other JAK inhibitors,and particularly other JAK inhibitors having a JAK affinity, selectivityor binding site different from CYT-0387. These properties can bedetermined using the JAK2 crystal structure and the modeling approachand activity assays described in U.S. Pat. No. 7,593,820, the entiredisclosure of which is incorporated herein by reference. To receive thefurther benefit resulting from subsequent CYT-0387 therapy, patientswill either be undergoing treatment with the other JAK2 inhibitor orwill have been treated with such a drug within a time frame, relative toCYT-0387 therapy onset, sufficient for the effects of that JA 2inhibitor to be manifest in the patient.

Patients selected for CYT-0387 therapy also include patients havingaltered levels of detectable protein markers. More particularly,patients in whom the levels of certain protein markers, includingcertain cytokines and chemokines, are elevated can experiencesignificant benefit when treated with CYT-0387, in terms of their anemiaresponse and/or their spleen response to CYT-0387 therapy. Inembodiments, elevation in the level of one or more of the followingprotein markers signifies that the patient is a preferred candidate forCYT-0387 therapy:

(1) EGF, or epidermal growth factor, the mature form of which comprisesresidues 971-1023 of the sequence having Swiss-Prot designation P01133;

(2) TNF-α, or tumour necrosis factor alpha, the mature and soluble formof which comprises residues 77-233 of the sequence having Swiss-Protdesignation P01375;

(3) G-CSF, or granulocyte colony stimulating factor, the mature form ofwhich comprises residues 30-207 of the sequence having Swiss-Protdesignation P09919;

(4) IFN-α, or interferon alpha, comprises a family of subtypes themature forms of which are well known in the art;

(5) MIP-IP3, or macrophage inflammatory protein 1-beta (now known alsoas C—C motif chemokine 4, or CCL4), the mature form which compriseseither residues 24-92 or 26-92 of the sequence having Swiss-Protdesignation PI 3236;

(6) HGF, or hepatocyte growth factor, the mature forms of which arebased on the sequence having Swiss-Prot designation P14210, and includethe alpha chain having residues 32-494 and the beta chain havingresidues 495-728;

(7) MIG, or monokine induced by gamma interferon (now known also asCXCL9), is within the family of chemotactic cytokines, the mature formof which comprises residues 23-125 of the sequence having Swiss-Protdesignation Q07325;

(8) VEGF, or vascular endothelial growth factor A, the mature form ofwhich comprises residues 27-232 of the sequence having Swiss-Protdesignation PI 5692.

Patients presenting for CYT-0387 therapy experience a significant spleenresponse when they are selected initially based on an elevation in thelevel of any one or more of the markers noted above. An elevated levelis a level that is greater than the level in a normal subject.

Patients presenting for CYT-0387 therapy can also experience asignificant anemia response when they are selected initially based on adepression in the level of the protein eotaxin. This protein, known alsoas eosinophil chemotatic protein and comprising residues 24-97 of thesequence having Swiss-Prot designation P51671, functions throughinteraction with CC 3 to promote accumulation of esoinophils in responseto allergens, a prominent feature of allergic inflammatory reactions.

Still other markers useful to select patients for CYT-0387 therapyinclude altered levels of EPO, hepcidin and BMP-2. Another example ofmarkers useful to select patients for CYT-0387 therapy includes thelevels of BMP-6.

Other markers may be used in monitoring CYT-0387 therapy or dosingregimen. By way of example, such markers may include but are not limitedto Compound 3, Compound 4, Compound 8, Compound 10, Compound 12, andCompound 13. The levels of these markers may be detected by the methodsthat are commonly used, such as those described in the examples of thepresent application.

The “level” of a given marker is considered to be altered, i.e., eitherelevated or reduced, when the level measured in a given patient isdifferent to a statistically significant extent from the correspondinglevel in a normal subject. Patients that present with marker levelsaltered to an extent sufficient, desirably, to yield a p value of atleast 0.05 or more significant, i.e., better, are selected as candidatesfor CYT-0387 therapy. In embodiments, the p value is at least 0.03, 0.02or 0.01, and in preferred embodiments the p value is at least 0.009,0.007, 0.005, 0.003, 0.001 or better.

The levels of a given marker can be determined using assays already wellestablished for detection the markers noted above. In embodiments, thisis achieved by extracting a biological sample from the patientcandidate, such as a sample of whole blood or a fraction thereof such asplasma or serum. The sample then is treated to enrich for the marker ofinterest, if desired, and the enriched or neat sample is assayed forinstance using a detectable ligand for the marker, such as a labeledantibody that binds selectively to the marker. The amount of markerpresent in the sample can then be determined either semi-quantitativelyor quantitatively, to obtain a value that is then compared against areference value that is the normal level for that marker in a healthysubject. As noted above, a difference in marker levels sufficient toarrive at a p value that is at least 0.05 indicates an altered markerlevel of significance, and patients presenting with an elevated level ofthat marker (or in the case of eotaxin, a decreased level) arecandidates for CYT-0387 therapy.

Also suitable as candidates for CYT-0387 therapy are those patients thatmeet certain clinical criteria, including those presenting with a spleenof relatively small size, and those presenting with an elevated level ofcirculating, or peripheral, blasts. These patients respond to CYT-0387therapy particularly well, in terms of their spleen response. In oneembodiment, the selected patient is one that has not yet progressed totransfusion dependency. Splenic enlargement is assessed by palpation.Splenic size and volume can also be measured by diagnostic imaging suchas ultrasound, CT or MRI). Normal spleen size is approximately 1 1.0 cm.in craniocaudal length.

Also suitable as candidates for CYT-0387 therapy are those patientspresenting with a lower percentage of circulating blasts. Blasts areimmature precursor cells that are normally found in the bone marrow andnot the peripheral blood. They normally give rise to mature blood cells.The lower percentage of circulating blasts is measured bycytomorphologic analysis of a peripheral blood smear as well asmultiparameter flow cytometry and immunohistochemistry. As a prognosticfactor>/=1% blasts is used.

The compounds according to the present application may be used incombination with one or more additional therapeutic agents. Thetherapeutic agents may be in the forms of compounds, antibodies,polypeptides, or polynucleotides. The therapeutic agent includes, but isnot limited to, a chemotherapeutic agent, an immunotherapeutic agent, aradiotherapeutic agent, an anti-neoplastic agent, an anti-cancer agent,an anti-proliferation agent, an anti-fibrotic agent, an anti-angiogenicagent, a therapeutic antibody, or any combination thereof.

In one embodiment, the application provides a formulation describedherein and an additional therapeutic agent as a combined preparation forsimultaneous, separate or sequential use in therapy, e.g. a method oftreating a disease, disorder, or condition that is mediated by JAK. Thetherapeutic agents may be those that inhibit or modulate the activitiesof Bruton's tyrosine kinase, spleen tyrosine kinase, apoptosissignal-regulating kinase, Janus kinase, lysyl oxidase, lysyloxidase-like proteins, matrix metallopeptidase, bromodomain-containingprotein, adenosine A2B receptor, isocitrate dehydrogenase,serine/threonine kinase TPL2, discoidin domain receptor,serine/threonine-protein kinases, IKK, MEK, EGFR, histone deacetylase,protein kinase C, or any combination thereof. In certain embodiments,the therapeutic agent may be selected from a PI3K (including PI3Kγ,PI3Kδ, PI3Kβ, PI3Kα, and/or pan-PI3K) inhibitor, a JAK (Janus kinase,including JAK1, JAK2, and/or JAK3) inhibitor, a SYK (spleen tyrosinekinase) inhibitor, a BTK (Bruton's tyrosine kinase) inhibitor, an A2B(adenosine A2B receptor) inhibitor, an ACK (activated CDC kinase,including ACK1) inhibitor, an ASK (apoptosis signal-regulating kinase,including ASK1) inhibitor, Auroa kinase, a BRD (bromodomain-containingprotein, including BRD4) inhibitor, a Bcl (B-cell CLL/lymphoma,including Bcl-1 and/or Bcl-2) inhibitor, a CAK (CDK-activating kinase)inhibitor, a CaMK (calmodulin-dependent protein kinases) inhibitor, aCDK (cyclin-dependent kinases, including CDK1, 2, 3, 4, and/or 6)inhibitor, a CK (casein kinase, including CK1 and/or CK2) inhibitor, aDDR (discoidin domain receptor, including DDR1 and/or DDR2) inhibitor, aEGFR inhibitor, a FXR (farnesoid x receptor) inhibitor, a FAK (focaladhesion kinase) inhibitor, a GSK (glycogen synthase kinase) inhibitor,a HDAC (histone deacetylase) inhibitor, an IDO (indoleamine2,3-dioxygenase) inhibitor, an IDH (isocitrate dehydrogenase, includingIDH1) inhibitor, an IKK (1-Kappa-B kinase) inhibitor, a KDM5 (lysinedemethylase) inhibitor, a LCK (lymphocyte-specific protein tyrosinekinase) inhibitor, a LOX (lysyl oxidase) inhibitor, a LOXL (lysyloxidase like protein, including LOXL1, LOXL2, LOXL3, LOXL4, and/orLOXL5) inhibitor, a MTH (mut T homolog) inhibitor, a MEK(mitogen-activated protein kinase kinase) inhibitor, a matrixmetalloprotease (MMP, including MMP2 and/or MMP9) inhibitor, amitogen-activated protein kinases (MAPK) inhibitor, a PD-1 (programmedcell death protein 1) inhibitor, a PD-L1 (programmed death-ligand 1)inhibitor, a PDGF (platelet-derived growth factor) inhibitor, aphosphorylase kinase (PK) inhibitor, a PLK (polo-like kinase, includingPLK1, 2, 3) inhibitor, a protein kinase (PK, including protein kinase A,B, C) inhibitor, a STK (serine/threonine kinase) inhibitor, a STAT(signal transduction and transcription) inhibitor, aserine/threonine-protein kinase inhibitor, a TB3K (tank-binding kinase)inhibitor, a TLR (toll-like receptor modulators, including TLR-1, TLR-2,TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, TLR-10, TLR-11, TLR-12,and/or TLR-13) inhibitor, a TK (tyrosine kinase) inhibitor, a TPL2(serine/threonine kinase) inhibitor, a NEK9 inhibitor, an Abl inhibitor,a p38 kinase inhibitor, a PYK inhibitor, a PYK inhibitor, a c-Kitinhibitor, a NPM-ALK inhibitor, a Flt-3 inhibitor, a c-Met inhibitor, aKDR inhibitor, a TIE-2 inhibitor, a VEGFR inhibitor, a SRC inhibitor, aHCK inhibitor, a LYN inhibitor, a FYN inhibitor, a YES inhibitor, achemotherapeutic agent, an immunotherapeutic agent, a radiotherapeuticagent, an anti-neoplastic agent, an anti-cancer agent, ananti-proliferation agent, an anti-fibrotic agent, an anti-angiogenicagent, a therapeutic antibody, or any combination thereof. In someembodiments, the PI3K-6 inhibitor is(S)-2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-oneas named by ChemDraw (may also be referred to as5-Fluoro-3-phenyl-2-[(1S)-1-(9H-purin-6-ylamino)propyl]quinazolin-4(3H)-one)and may be synthesized by the methods described in U.S. Pat. No.7,932,260. In certain embodiment, the SyK inhibitor is6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amineas named by ChemDraw (may also be referred to as6-(1H-indazol-6-yl)-N-[4-(morpholin-4-yl)phenyl]imidazo[1,2-a]pyrazin-8-amine)and may be synthesized by the methods described in U.S. Pat. No.8,450,321. In other embodiments, the BTK inhibitor is(S)-6-amino-9-(1-(but-2-ynoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-8(9H)-oneas named by ChemDraw (may also be6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one)and may be synthesized by the methods in U.S. Pat. No. 8,557,803.

Chemotherapeutic agents may be categorized by their mechanism of actioninto, for example, the following groups: anti-metabolites/anti-canceragents, such as pyrimidine analogs (floxuridine, capecitabine, andcytarabine); purine analogs, folate antagonists and related inhibitors,antiproliferative/antimitotic agents including natural products such asvinca alkaloid (vinblastine, vincristine) and microtubule such as taxane(paclitaxel, docetaxel), vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide); DNA damagingagents (actinomycin, amsacrine, busulfan, carboplatin, chlorambucil,cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin,doxorubicin, epirubicin, iphosphamide, melphalan, merchlorehtamine,mitomycin, mitoxantrone, nitrosourea, procarbazine, taxol, taxotere,teniposide, etoposide, triethylenethiophosphoramide); antibiotics suchas dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin),idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin; enzymes (L-asparaginase which systemicallymetabolizes L-asparagine and deprives cells which do not have thecapacity to synthesize their own asparagine); antiplatelet agents;antiproliferative/antimitotic alkylating agents such as nitrogenmustards cyclophosphamide and analogs, melphalan, chlorambucil), and(hexamethylmelamine and thiotepa), alkyl nitrosoureas (BCNU) andanalogs, streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,oxiloplatinim, carboplatin), procarbazine, hydroxyurea, mitotane,aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,goserelin, bicalutamide, nilutamide) and aromatase inhibitors(letrozole, anastrozole); anticoagulants (heparin, synthetic heparinsalts and other inhibitors of thrombin); fibrinolytic agents (such astissue plasminogen activator, streptokinase and urokinase), aspirin,dipyridamole, ticlopidine, clopidogrel; antimigratory agents;antisecretory agents (breveldin); immunosuppressives tacrolimussirolimus azathioprine, mycophenolate; compounds (TNP-470, genistein)and growth factor inhibitors (vascular endothelial growth factorinhibitors, fibroblast growth factor inhibitors); angiotensin receptorblocker, nitric oxide donors; anti-sense oligonucleotides; antibodies(trastuzumab, rituximab); cell cycle inhibitors and differentiationinducers (tretinoin); inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin, irinotecan and mitoxantrone, topotecan,irinotecan, camptothesin), corticosteroids (cortisone, dexamethasone,hydrocortisone, methylpednisolone, prednisone, and prenisolone); growthfactor signal transduction kinase inhibitors; dysfunction inducers,toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetellapertussis adenylate cyclase toxin, or diphtheria toxin, and caspaseactivators; and chromatin.

As used herein the term “chemotherapeutic agent” or “chemotherapeutic”(or “chemotherapy,” in the case of treatment with a chemotherapeuticagent) is meant to encompass any non-proteinaceous (i.e, non-peptidic)chemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; emylerumines and memylamelamines includingalfretamine, triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimemylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingsynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (articularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, foremustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicin phill,see, e.g., Agnew, Chem. Intl. Ed. Engl, 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carrninomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK(r); razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g., paclitaxel (TAXOL(r) and docetaxel (TAXOTERE(r)); chlorambucil;gemcitabine (Gemzar(r)); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vancristine;vinorelbine (Navelbine(r)); novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin, andirinotecan) and pharmaceutically acceptable salts, acids or derivativesof any of the above. One or more chemotherapeutic agent are used orincluded in the present application. For example, gemcitabine,nab-paclitaxel, and gemcitabine/nab-paclitaxel are used with the JAKinhibitor and/or PI3Kδ inhibitor for treating hyperproliferativedisorders.

Also included in the definition of “chemotherapeutic agent” areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including Nolvadex™),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston(r)); inhibitors of theenzyme aromatase, which regulates estrogen production in the adrenalglands, such as, for example, 4(5)-imidazoles, aminoglutethimide,megestrol acetate (Megace(r)), exemestane, formestane, fadrozole,vorozole (Rivisor(r)), letrozole (Femara(r)), and anastrozole(Arimidex(r)); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprohde, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The anti-angiogenic agents include, but are not limited to, retinoidacid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN(r),ENDOSTATIN(r), suramin, squalamine, tissue inhibitor ofmetalloproteinase-1, tissue inhibitor of metalloproternase-2,plasminogen activator inhibitor-1, plasminogen activator inhibitor-2,cartilage-derived inhibitor, paclitaxel (nab-paclitaxel), plateletfactor 4, protamine sulphate (clupeine), sulphated chitin derivatives(prepared from queen crab shells), sulphated polysaccharidepeptidoglycan complex (sp-pg), staurosporine, modulators of matrixmetabolism, including for example, proline analogs((1-azetidine-2-carboxylic acid (LACA), cishydroxyproline,d,I-3,4-dehydroproline, thiaproline, .alpha.-dipyridyl,beta-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone; methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin,beta-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodiumthiomalate, d-penicillamine (CDPT), beta-1-anticollagenase-serum,alpha-2-antiplasmin, bisantrene, lobenzarit disodium,n-2-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”,thalidomide; angiostatic steroid, cargboxynaminolmidazole;metalloproteinase inhibitors such as BB94. Other anti-angiogenesisagents include antibodies, preferably monoclonal antibodies againstthese angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, VEGFisoforms, VEGF-C, HGF/SF and Ang-1/Ang-2. See Ferrara N. and Alitalo, K.“Clinical application of angiogenic growth factors and their inhibitors”(1999) Nature Medicine 5:1359-1364.

The anti-fibrotic agents include, but are not limited to, the compoundssuch as beta-aminoproprionitrile (BAPN), as well as the compoundsdisclosed in U.S. Pat. No. 4,965,288 to Palfreyman, et al., issued Oct.23, 1990, entitled “Inhibitors of lysyl oxidase,” relating to inhibitorsof lysyl oxidase and their use in the treatment of diseases andconditions associated with the abnormal deposition of collagen; U.S.Pat. No. 4,997,854 to Kagan, et al., issued Mar. 5, 1991, entitled“Anti-fibrotic agents and methods for inhibiting the activity of lysyloxidase in situ using adjacently positioned diamine analogue substrate,”relating to compounds which inhibit LOX for the treatment of variouspathological fibrotic states, which are herein incorporated byreference. Further exemplary inhibitors are described in U.S. Pat. No.4,943,593 to Palfreyman, et al., issued Jul. 24, 1990, entitled“Inhibitors of lysyl oxidase,” relating to compounds such as2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine; as well as, e.g.,U.S. Pat. No. 5,021,456; U.S. Pat. No. 5,5059,714; U.S. Pat. No.5,120,764; U.S. Pat. No. 5,182,297; U.S. Pat. No. 5,252,608 (relating to2-(1-naphthyloxymemyl)-3-fluoroallylamine); and U.S. Patent ApplicationNo. 2004/0248871, which are herein incorporated by reference. Exemplaryanti-fibrotic agents also include the primary amines reacting with thecarbonyl group of the active site of the lysyl oxidases, and moreparticularly those which produce, after binding with the carbonyl, aproduct stabilized by resonance, such as the following primary amines:emylenemamine, hydrazine, phenylhydrazine, and their derivatives,semicarbazide, and urea derivatives, aminonitriles, such asbeta-aminopropionitrile (BAPN), or 2-nitroethylamine, unsaturated orsaturated haloamines, such as 2-bromo-ethylamine, 2-chloroethylamine,2-trifluoroethylamine, 3-bromopropylamine, p-halobenzylamines,selenohomocysteine lactone. Also, the anti-fibrotic agents are copperchelating agents, penetrating or not penetrating the cells. Exemplarycompounds include indirect inhibitors such compounds blocking thealdehyde derivatives originating from the oxidative deamination of thelysyl and hydroxylysyl residues by the lysyl oxidases, such as thethiolamines, in particular D-penicillamine, or its analogues such as2-amino-5-mercapto-5-methylhexanoic acid,D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphurate,2-acetamidoethyl-2-acetamidoethanethiol sulphanate,sodium-4-mercaptobutanesulphinate trihydrate.

The immunotherapeutic agents include and are not limited to therapeuticantibodies suitable for treating patients; such as abagovomab,adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab,anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab,bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab,cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab,daratumumab, drozitumab, duligotumab, dusigitumab, detumomab,dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab,ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab,flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab,glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab,inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab,lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab,matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab,narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomabn,ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab,oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab,pertuzumab, pintumomab, pritumumab, racotumomab, radretumab,rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab,siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab,tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab,tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab,zalutumumab, obinutuzumab, CC49 and 3F8. The exemplified therapeuticantibodies may be further labeled or combined with a radioisotopeparticle, such as indium In 111, yttrium Y 90, iodine I-131.

The application also provides method for treating a subject who isundergoing one or more standard therapies, such as chemotherapy,radiotherapy, immunotherapy, surgery, or combination thereof.Accordingly, one or more therapeutic agent or inhibitors may beadministered before, during, or after administration of chemotherapy,radiotherapy, immunotherapy, surgery or combination thereof.

Other examples of chemotherapy treatments (including standard orexperimental chemotherapies) are described below. In addition, treatmentof certain lymphomas is reviewed in Cheson, B. D., Leonard, J. P.,“Monoclonal Antibody Therapy for B-Cell Non-Hodgkin's Lymphoma” The NewEngland Journal of Medicine 2008, 359(6), p. 613-626; and Wierda, W. G.,“Current and Investigational Therapies for Patients with CLL” Hematology2006, p. 285-294. Lymphoma incidence patterns in the United States isprofiled in Morton, L. M., et al. “Lymphoma Incidence Patterns by WHOSubtype in the United States, 1992-2001” Blood 2006, 107(1), p. 265-276.

Examples of immunotherapeutic agents include, but are not limited to,rituximab (such as Rituxan), alemtuzumab (such as Campath, MabCampath),anti-CD19 antibodies, anti-CD20 antibodies, anti-MN-14 antibodies,anti-TRAIL, Anti-TRAIL DR4 and DR5 antibodies, anti-CD74 antibodies,apolizumab, bevacizumab, CHIR-12.12, epratuzumab (hLL2-anti-CD22humanized antibody), galiximab, ha20, ibritumomab tiuxetan, lumiliximab,milatuzumab, ofatumumab, PRO131921, SGN-40, WT-1 analog peptide vaccine,WT1 126-134 peptide vaccine, tositumomab, autologous human tumor-derivedHSPPC-96, and veltuzumab. Additional immunotherapy agents includes usingcancer vaccines based upon the genetic makeup of an individual patient'stumor, such as lymphoma vaccine example is GTOP-99 (MyVax®).

Examples of chemotherapy agents include aldesleukin, alvocidib,antineoplaston AS2-1, antineoplaston A10, anti-thymocyte globulin,amifostine trihydrate, aminocamptothecin, arsenic trioxide, betaalethine, Bcl-2 family protein inhibitor ABT-263, ABT-199, BMS-345541,bortezomib (Velcade®), bryostatin 1, busulfan, carboplatin, campath-1H,CC-5103, carmustine, caspofungin acetate, clofarabine, cisplatin,Cladribine (Leustarin), Chlorambucil (Leukeran), Curcumin, cyclosporine,Cyclophosphamide (Cyloxan, Endoxan, Endoxana, Cyclostin), cytarabine,denileukin diftitox, dexamethasone, DT PACE, docetaxel, dolastatin 10,Doxorubicin (Adriamycin®, Adriblastine), doxorubicin hydrochloride,enzastaurin, epoetin alfa, etoposide, Everolimus (RAD001), fenretinide,filgrastim, melphalan, mesna, Flavopiridol, Fludarabine (Fludara),Geldanamycin (17-AAG), ifosfamide, irinotecan hydrochloride,ixabepilone, Lenalidomide (Revlimid®, CC-5013), lymphokine-activatedkiller cells, melphalan, methotrexate, mitoxantrone hydrochloride,motexafin gadolinium, mycophenolate mofetil, nelarabine, oblimersen(Genasense) Obatoclax (GX15-070), oblimersen, octreotide acetate,omega-3 fatty acids, oxaliplatin, paclitaxel, PD0332991, PEGylatedliposomal doxorubicin hydrochloride, pegfilgrastim, Pentstatin (Nipent),perifosine, Prednisolone, Prednisone, R-roscovitine (Selicilib, CYC202),recombinant interferon alfa, recombinant interleukin-12, recombinantinterleukin-11, recombinant flt3 ligand, recombinant humanthrombopoietin, rituximab, sargramostim, sildenafil citrate,simvastatin, sirolimus, Styryl sulphones, tacrolimus, tanespimycin,Temsirolimus (CCl-779), Thalidomide, therapeutic allogeneic lymphocytes,thiotepa, tipifarnib, Velcade® (bortezomib or PS-341), Vincristine(Oncovin), vincristine sulfate, vinorelbine ditartrate, Vorinostat(SAHA), vorinostat, and FR (fludarabine, rituximab), CHOP(cyclophosphamide, doxorubicin, vincristine, prednisone), CVP(cyclophosphamide, vincristine and prednisone), FCM (fludarabine,cyclophosphamide, mitoxantrone), FCR (fludarabine, cyclophosphamide,rituximab), hyperCVAD (hyperfractionated cyclophosphamide, vincristine,doxorubicin, dexamethasone, methotrexate, cytarabine), ICE(iphosphamide, carboplatin and etoposide), MCP (mitoxantrone,chlorambucil, and prednisolone), R-CHOP (rituximab plus CHOP), R-CVP(rituximab plus CVP), R-FCM (rituximab plus FCM), R-ICE (rituximab-ICE),and R-MCP (R-MCP).

The therapeutic treatments can be supplemented or combined with any ofthe abovementioned therapies with stem cell transplantation ortreatment. One example of modified approach is radioimmunotherapy,wherein a monoclonal antibody is combined with a radioisotope particle,such as indium In 111, yttrium Y 90, iodine 1-131. Examples ofcombination therapies include, but are not limited to, Iodine-131tositumomab (Bexxar®), Yttrium-90 ibritumomab tiuxetan (Zevalin®),Bexxar® with CHOP.

Other therapeutic procedures include peripheral blood stem celltransplantation, autologous hematopoietic stem cell transplantation,autologous bone marrow transplantation, antibody therapy, biologicaltherapy, enzyme inhibitor therapy, total body irradiation, infusion ofstem cells, bone marrow ablation with stem cell support, invitro-treated peripheral blood stem cell transplantation, umbilical cordblood transplantation, immunoenzyme technique, pharmacological study,low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery,radiation therapy, and nonmyeloablative allogeneic hematopoietic stemcell transplantation.

It is understood that the below examples illustrate certain aspects ofthe present application. It is also understood that values andparameters shown in the examples may be modified within reasonablevariation, and that various modifications may be made within the scopeof the present application.

Example 1 Methods of Making

N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) can be synthesized as described in U.S. Pat. No. 8,486,941and PCT Application WO 2012/071612.

CYT-0387 Dihydrochloride Monohydrate Form II from CYT-0387Dihydrochloride Anhydrous Form I

To a suspension ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride anhydrous Form I in methanol was added amolar excess of hydrochloric acid in water. The resulting solids wereisolated and washed with methanol and aqueous hydrochloric acid to yieldCYT-0387 dihydrochloride monohydrate Form II.

CYT-0387 Dihydrochloride Monohydrate Form II from CYT-0387 Free Base

To a suspension ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) free base in methanol was added a molar excess ofconcentrated hydrochloric acid. The resulting suspension was optionallyseeded withN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride monohydrate Form II and water was added. Theoptionally seededN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride monohydrate Form II may be prepared asdescribed above. The resulting solids were isolated and washed withmethanol and aqueous hydrochloric acid to yield CYT-0387 dihydrochloridemonohydrate Form II.

CYT-0387 Monohydrochloride Anhydrous Form I from CYT-0387 Free Base

To a suspension ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) free base in methanol was added 1.0 molar equivalents ofconcentrated hydrochloric acid. The resulting solids were isolated andwashed with methanol to yield CYT-0387 monohydrochloride anhydrous FormI.

CYT-0387 Monohydrochloride Anhydrous Form III from CYT-0387Monohydrochloride Anhydrous Form I

A suspension of CYT-0387 monohydrochloride anhydrous Form I is stirredin water/THF (30% water; v/v). The resulting solids were isolated andwashed with a water/THF mixture to yield CYT-0387 monohydrochlorideanhydrous Form III.

CYT-0387 Monohydrochloride Anhydrous Form III from CYT-0387Dihydrochloride Monohydrate Form II

A suspension of CYT-0387 dihydrochloride monohydrate Form II is stirredin methanol/water (30% water; v/v). The resulting solids were isolatedand washed with a methanol/water mixture to yield CYT-0387monohydrochloride anhydrous Form III.

The above forms were characterized by various analytical techniques,including X-ray powder diffraction pattern (XPPD), differential scanningcalorimetry (DSC), thermographic analysis (TGA), and dynamic vaporsorption (DVS) using the procedures described below.

X-Ray Powder Diffraction:

XRPD analysis was conducted on a diffractometer (PANanalyticalXPERT-PRO, PANanalytical B.V., Almelo, Netherlands) using copperradiation (Cu Kα, λ=1.5418 Å). Samples were prepared for analysis bydepositing the powdered sample in the center of an aluminum holderequipped with a zero background plate. The generator was operated at avoltage of 45 kV and amperage of 40 mA. Slits used were Soller 0.02rad., antiscatter 1.00, and divergence. The sample rotation speed was 2sec. Scans were performed from 2 to 400 2θ during 15 min with a stepsize of 0.0167° 2θ. Data analysis was performed by X'Pert Highscoreversion 2.2c (PANalytical B.V., Almelo, Netherlands) and X'Pert dataviewer version 1.2d (PANalytical B.V., Almelo, Netherlands).

The XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II is represented in FIG. 5.

The XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I is represented in FIG. 6.

The XRPD pattern forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III is represented in FIG. 7.

XRPD peaks of the various CYT-0387 forms are found in Table 1 above.

Differential Scanning Calorimetry:

Thermal properties ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II were evaluated using a DifferentialScanning Calorimetry (DSC) instrument (TA Q1000, TA Instruments, NewCastle, Del., USA). Approximately 5 to 10 mg of solid sample was placedin a standard aluminum pan vented with a pinhole for each experiment andheated at a rate of 10° C./min under a 50 mL/min nitrogen purge. Dataanalysis was conducted using Universal Analysis 2000 Version 4.7A (TAInstruments, New Castle, Del., USA). Heat of fusion analysis wasconducted by sigmoidal integration of the endothermic melting peak.

The DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II is represented in FIG. 8.

The DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I is represented in FIG. 9.

The DSC forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III is represented in FIG. 10.

Thermogravimetric analysis: Thermogravimetric analysis (TGA) ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II was performed on a TGA instrument(TA Q500, TA Instruments, New Castle, Del., USA). Approximately 5 to 10mg of solid sample was placed in an open aluminum pan for eachexperiment and heated at a rate of 10° C./min under a 60 mL/min nitrogenpurge using. Data analysis was conducted using Universal Analysis 2000Version 4.7A (TA Instruments, New Castle, Del., USA).

The TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II is represented in FIG. 11.

The TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I is represented in FIG. 12.

The TGA forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III is represented in FIG. 13.

Dynamic vapor sorption: The hygroscopicity ofN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II was evaluated at room temperatureusing a dynamic vapor sorption (DVS) instrument (TGA Q5000 TAInstruments, New Castle, Del.). Water adsorption and desorption werestudied as a function of relative humidity (RH) over the range of 0 to90% at room temperature. The humidity in the chamber was increased fromthe initial level 50% RH) to 60% RH and held until the solid andatmosphere reached equilibration. The equilibrium test was continueduntil passed or expired after 10 hours. At this point, RH was raised 10%higher and the process was repeated until 90% RH was reached andequilibrated. During this period, the water sorption was monitored. Fordesorption, the relative humidity was decreased in a similar manner tomeasure a full sorption/desorption cycle. All experiments were operatedin dm/dt mode (mass variation over time) to determine the equilibrationendpoint. Approximately 4 mg of solid CYT-0387 was used. Data analysiswas conducted using Universal Analysis 2000 Version 4.7A (TAInstruments, New Castle, Del., USA).

The DVS forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II is represented in FIG. 14.

The single crystal X-ray crystallography data forN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide(CYT-0387) dihydrochloride monohydrate Form II andN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamideCYT-0387) monohydrochloride anhydrous Form I is summarized in Table 2below. Data from further characterization of the crystals are summarizedin Table 3 below.

TABLE 2 Single Crystal X-ray Crystallography Data Unit Cell DimensionsForm and Composition Distance (Å) Angle (°) Form API:water:solvent a b cα β γ CYT-0387 1:1:0 10.2837(6) 10.4981(6) 11.5143(7) 83.297(2)87.649(2) 67.445(2) dihydro- chloride monohydrate Form II CYT-0387 1:0:09.4255(3) 11.6729(4) 19.7561(6) 85.3940(10) 88.103(2) 83.821(2)monohydro- chloride anhydrous Form I

TABLE 3 Crystal Data and Structure Refinement CYT-0387 CYT-0387dihydrochloride monohydrochloride Property monohydrate Form II anhydrousForm I Empirical formula C₂₃H₂₆Cl₂N₆O₃ C₂₃H₂₃ClN₆O₂ Formula weight505.40 450.92 Temperature 100(2) K 100(2) K Wavelength 1.54178 Å 1.54178Å Crystal system Triclinic Triclinic Space group P-1 P-1 Volume1140.14(12) Å³ 2153.36(12) Å³ Z 2  4  Density (calculated) 1.472 g/cm³1.391 g/cm³ 100(2) K represents 100 ± 2° K

Microscopic images of the various forms of the invention were acquiredusing an Olympus polarizing microscope (BX-51, Olympus, Center Valley,Pa., USA). The samples were dispersed in mineral oil and examined undercross-polarized light using a 530 nm wave plate (results not shown).

Example 2

Tablets comprising CYT-0387 dihydrochloride monohydrate Form II inamounts equivalent to 100 mg, 150 mg, and 200 mg of CYT-0387 free basecan be prepared according to the process described herein. Tabletscomprising CYT-0387 dihydrochloride monohydrate Form II in amountsequivalent to 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg of CYT-0387free base were prepared. The below Table 4, summarizes the formulationsof such tablets.

TABLE 4 Tablet Formulation Quantity per Dosage Unit 100 mg 150 mg 200 mg250 mg 300 mg Amount Strength Strength strength strength strengthComponent (% w/w) (mg) (mg) (mg) (mg) (mg) CYT-0387 40.65 121.94 182.91243.88 304.88 365.85 Dihydrochloride Monohydrate Form II Propyl Gallate0.20 0.60 0.90 1.20 1.50 1.80 Microcrystalline 34.23 102.70 154.05205.40 256.73 308.07 Cellulose (PH 105) Lactose (Fast Flo 316) 16.9250.76 76.14 101.52 126.9 152.28 Sodium Starch 6.00 18.00 27.00 36.0045.00 54.00 Glycolate Colloidal Silicon 0.50 1.50 2.25 3.00 3.75 4.50Dioxide Magnesium Stearate 1.50* 4.50 6.750 9.00 11.25 13.50 Core Tablet100.00 300.00 450.00 600.00 750.00 900.00 Total Opadry II Brown 4.0012.00 18.00 24.00 30.00 36.00 85F165010 Film-Coated Tablet 104.00 312.00468.00 624.00 780.00 936.00 Total *0.75% intragranular, 0.75%extragranular Mg stearate

The tablet formulation of Table 4 comprised propyl gallate, whichdecreased the extent or levels of oxidative degradation of CYT-0387 FormII and resulted in increased stability of CYT-0387 Form II. This wasdetermined from the study that investigated the potential effects ofdifferent antioxidants in inhibiting or preventing degradation ofCYT-0387 dihydrochloride monohydrate Form II. In the initial study, fiveantioxidants of three different mechanisms of action were examined: freeradical scavenger antioxidants (propyl gallate, butylated hydroxyanisole(BHA), and butylated hydroxytoluene (BHT)), a sacrificial reductant(ascorbic acid), and an oxygen scavenger (sodium metabisulfite).

Aqueous solutions containing 20 g/mL of CYT-0387 dihydrochloridmonohydrate Form II in 70% (v/v) 50 mM acetate buffer (pH 4.0) and 30%(v/v) methanol were incubated in absence (which was used as control) andpresence of 0.1% (w/v) antioxidant at 60° C. for up to 7 days. At Day 0,5 and 7, the solution was analyzed using a reverse-phase HPLC with aZorbax SB-C8 column (Phenomenex, Torrance, Calif.). As shown in Table 7,the presence of propyl gallate, BHA, or ascorbic acid, at the level of0.1% (w/v), inhibited or prevented the degradation of CYT-0387 Form IIrelative to BHT and sodium metabisulfite at the same level. Less thanone percent of CYT-0387 Form II was degraded in the presence of propylgallate or BHA. While total degradation products could not be determinedfor 0.1% ascorbic acid due to interference, the main oxidativedegradation products were not observed (data not shown).

In additional studies, the effects of the lower concentrations of 0.01%and 0.001% (w/v) of propyl gallate, BHA, and ascorbic acid on thestability of CYT-0387 were examined under the same condition. Theresults from these studies are summarized in Table 7. The results showedthat, at 0.01% (w/v) antioxidant level, increased stability of CYT-0387Form II was observed in the presence of propyl gallate or ascorbic acidat 60° C. for up to 7 days. Moreover, at 0.001% (w/v) antioxidant level,the presence of propyl gallate decreased or inhibited the degradation ofCYT-0387 relative to BHA and ascorbic acid (Table 7). These resultsindicated that, among the tested antioxidants, propyl gallate was themost effective in inhibiting or preventing the degradation of CYT-0387dihydrochloride monohydrate Form II.

TABLE 7 Effects of antioxidants on degradation of CYT- 0387 Form II inpH 4 aqueous buffer at 60° C. Time Total Amount Point Degradation^(a)Antioxidant (% w/v) (Days) (%) Control 0 0 0.29 5 17.24 7 21.84Butylated 0.1 0 0.06 hydroxytoluene 5 1.71 (BHT) 7 4.35 Sodium 0.1 00.33 Metabisulfite 5 43.83 7 46.31 Propyl gallate 0.1 0 0.29 5 0.31 70.32 0.01 0 0.22 5 0.38 7 0.72 0.001 0 0.22 5 1.98 7 4.32 Butylated 0.10 0.45 hydroxyanisole 5 0.50 (BHA) 7 1.24 0.01 0 0.55 5 1.75 7 3.040.001 0 0.30 5 7.64 7 13.76 Ascorbic Acid 0.1 0 N/A^(b) 5 N/A^(b) 7N/A^(b) 0.01 0 0.20 5 1.58 7 1.78 0.001 0 0.51 5 17.95 7 21.28^(a)Include impurity that was present in the formulation ^(b)N/A: Notavailable

Next, the stability of 100 mg tablet formulation comprising CYT-0387Form II was examined in the presence of 0%, 0.2%, 0.5%, or 1.0% propylgallate at 25° C./60% RH (relative humidity) or 40° C./75% RH for up to6 months. The degradation profiles were determined at month 0, 1, 3, and6. Results of the studies at 40° C./75% RH for up to 6 months weresummarized in Table 8. The results showed that, at 40° C./75% RH,CYT-0387 Form II tablet formulation, at 100 mg, having 0.2% propylgallate exhibited increased stability compared to CYT-0387 Form IItablet formulation, at 100 mg, having 0%, 0.5% or 1.0% propyl gallate.The results of the study at 25° C./60% RH showed that the degradation ofCYT-0387 Form II was also reduced by propyl gallate at 0.2%, 0.5%, and1.0% (data not shown). The observed trend was similar for thedegradation profiles at 25° C./60% RH as that observed at 40° C./75% RH,i.e. increased stability of CYT-0387 Form II in the tablet formulationwas observed in 0.2% propyl gallate compared to 0%, 0.5%, and 1% propylgallate (data not shown). Taken together, these results indicate that,among the anti-oxidants and the percentages that were examined in thesestudies, propyl gallate at 0.2% provided the optional level of stabilityof CYT-0387 dihydrochloride monohydrate Form II.

TABLE 8 Effects of levels of propyl gallate on degradation of CYT-0387Form II in the 100 mg tablet formulation. Propyl Time Total GallatePoint Degradation (%) (Months) (%) 0 0 0.10 1 0.68 3 1.01 6 1.28 0.2 00.10 1 0.34 3 0.67 6 0.80 0.5 0 0.11 1 0.39 3 0.67 6 0.95 1.0 0 0.14 10.44 3 0.87 6 1.34

Example 3

Tablets comprising CYT-0387 dihydrochloride monohydrate Form II (dosesequivalent to 100, 150, 200, and 300 mg of the free base) and capsulescomprising CYT-0387 dihydrochloride anhydrous Form I (dose equivalent to300 mg of the free base) were evaluated in a Phase 1, single dose studyin healthy subjects.

Intensive PK and PD sampling occurred from 0.5 hour up to 36 hours postdose. Safety was monitored throughout the study. A parametric analysisof variance (ANOVA) using a mixed-effects model was used to fit to thenatural logarithmic transformation of PK parameters (AUC and C_(max)).The 90% confidence intervals were constructed for the ratio of geometricmeans of CYT-0387 dihydrochloride monohydrate Form II tablets at 100,150, 200 and 300 mg vs CYT-0387 dihydrochloride anhydrous Form I capsuleat 300 mg, using equivalence bounds of 70% to 143% for AUC and C_(max).The pharmacokinetic data is represented in Table 5.

TABLE 5 Pharmacokinetic data of CYT-0387 dihydrochloride monohydrateForm II tablet and CYT-0387 dihydrochloride anhydrous Form I capsuleformulations CYT-0387 CYT-0387 dihydrochloride dihydrochloride anhydrousmonohydrate Form I Form II Plasma PK Tablet: 306 mg capsule GMR (%)Tablet Dose Parameters Mean (SB) Mean (SD) (90% CI) 100 mg AUC_(inf) 1360 (497.9) 2813 (1984) 55.8 (42.1, 73.9) (h · ng/mL) C_(max) (ng/mL)166.5 (73.3)  388.8 (225.0) 47.3 (35.4, 63.2) 150 mg AUC_(inf) 3018(1532) 4285 (1923) 69.9 (59.6, 81.9) (h · ng/mL) C_(max) (ng/mL) 354.8(150.6) 549.3 (259.9) 65.6 (55.2, 77.8) 200 mg AUC_(inf) 2572 (1671)2672 (1993) 101.9 (87.7, 118.4) (h · ng/mL) C_(max) (ng/mL) 323.7(188.3) 356.1 (195.6)  92.0 (79.0, 107.1) 300 mg AUC_(inf) 3194 (1445)2586 (1481)  136.2 (107.5, 172.4) (h · ng/mL) C_(max) (ng/mL) 415.3(183.2) 381.8 (200.8) 115.7 (87.7, 152.7)

Example 4

CYT-0387 is a selective small molecule inhibitor of Janus kinase 1 and 2(JAK1/JAK2) currently under investigation to treat myelofibrosis. Thisstudy evaluated the mass balance/recovery, metabolite profile,pharmacokinetics, and safety of radiolabeled CYT-0387 in humans.

Six healthy individuals (subjects) received a single oral dose of 200 mgCYT-0387 containing ˜100 μCi of [¹⁴C]-CYT-0387. Blood samples werecollected up to 21 days or until plasma radioactivity in 2 consecutivesamples was below detection limit or urine and feces sampling wasdiscontinued. Urine/feces samples were collected up to 21 days oruntil >90% administered dose was recovered in feces and urine andradioactivity in 2 consecutive sampling intervals were <1% administereddose. Plasma concentrations of CYT-0387 and metabolites were measuredusing LC-MS/MS and total radioactivity assessed by liquid scintillationcounting. Metabolite profiling was performed in select urine, feces, andplasma samples. Safety assessments were performed throughout the study.

Results:

CYT-0387 was well tolerated. No Grade 3 or 4 AEs, SAEs, or AEs leadingto study discontinuation were reported. The most frequently reported AEswere dizziness, headache, and nausea. Maximum concentration ofdrug-derived radioactivity in plasma was observed at 2.5 hours postdose.Mean blood-to-plasma concentration ratios ranged from 0.7 to 0.9 through24 hours postdose, indicating low association of radioactivity withblood cells. Overall recovery of radioactivity was 96.7% (feces: 69.3%;urine: 27.5%). The circulating radioactivity consisted mainly ofmetabolite M21 (64.2%), CYT-0387 (17.3%), and metabolites (M8: 5.8%;metabolite M19: 5.2%; M5: 2.7%; M28: 2.5%; and M20: 2.3%). The majorcomponent excreted in feces was M14 (21.4% of the dose), along withCYT-0387 (12.6% of the dose) and other metabolites (M21: 12.7% of thedose; co-eluted M19/M33: 7.1% of the dose). The remaining identified 10metabolites in feces each accounted for less than 5% of the dose. Inurine, metabolite M21 was the main species (11.5% of the dose), with lowlevels of minor metabolites observed.

After oral administration in healthy subjects, [¹⁴C]-CYT-0387 wasprimarily eliminated in the feces, as a combination of metabolites andunchanged parent drug.

Example 5

CYT-0387 is a selective small molecule inhibitor of Janus kinase 1 and 2(JAK1/JAK2), and is currently under investigation to treatmyelofibrosis. In a Phase 1/2 study in myelofibrosis patients, 300 mgCYT-0387 capsule once daily was selected as Phase 3 dose based on afavorable benefit:risk profile. An immediate release tablet formulation(CYT-0387 tablet) was developed for further clinical evaluation. Therelative bioavailability of CYT-0387 tablet vs capsule was evaluated inthis study to identify Phase 3 dose of CYT-0387 tablet. CYT-0387 tablet(100 to 300 mg) vs capsule (300 mg) pharmacokinetics (PK) after a singledose was evaluated in healthy subjects. CYT-0387 tablet PK atsupratherapeutic doses (400 and 800 mg), under fed and fastedconditions, and with an acid reducing agent (i.e., omeprazole) was alsoevaluated. Intensive PK sampling occurred up to 36 hours postdose.Safety was monitored throughout the study. A parametric analysis ofvariance (ANOVA) using a mixed-effects model was used to fit to thenatural logarithmic transformation of PK parameters (AUC and C_(max)).The 90% confidence intervals were constructed for the ratio of geometricmeans of CYT-0387 tablet PK at 100, 150, 200 and 300 mg vs CYT-0387capsule 300 mg, using equivalence bounds of 70% to 143% for AUC andC_(max). A similar approach was used to assess the effect of food andomeprazole.

CYT-0387 tablet at 200 mg provided plasma exposures equivalent toCYT-0387 capsule at 300 mg (Table 6). CYT-0387 plasma exposuresincreased less-than-dose-proportionally from 100 to 800 mg. Intake oflight and high-fat meal modestly increased C_(max) (38% and 28% increasefor light- and high-fat meals, respectively) and AUC_(inf) (16% and 28%increase for light- and high-fat meals, respectively) for CYT-0387tablet. Omeprazole reduced the exposure of CYT-0387 tablet by 36% forC_(max) and 33% for AUC_(inf). These differences were not consideredclinically relevant.

CYT-0387 tablet at 200 mg provides comparable exposure to CYT-0387capsule at 300 mg. CYT-0387 tablet plasma exposures increased in aless-than-dose-proportional manner. No clinically relevant effects offood or acid reducing agents were observed on CYT-0387 tablet PK.

TABLE 6 Relative bioavailability of CYT-0387 phase 3 tablet vs capsulefollowing single dose CYT-0387 administration PK 200 mg Tablet: 300 mgCapsule: Parameters Mean (% CV) Mean (% CV) GMR [%] (90% CI) C_(max) 323.7 (58.2)  356.1 (54.9)  91.99 (78.98, 107.15) (ng/mL) AUC_(inf)2549.7 (66.1) 2665.5 (74.9) 101.69 (87.45, 118.26) (h · ng/mL)AUC_(last) 2324.5 (65.3) 2443.9 (71.3) 100.35 (86.73, 116.10) (h ·ng/mL) % CV = % coefficient of variation; CI = confidence interval; datarounded as applicable and shown as three significant figures

Example 6

This example described the preparation of M14 (Compound 3), M8 (Compound4), M20 (Compound 12), M21 (Compound 13), Compound 8 and Compound 10.

To a flask was charged 4-(2-chloropyrimidin-4-yl)benzoic acid (3.0 g,12.8 mmol), 4-morpholonoaniline (2.7 g, 14.0 mmol, 1.1 equiv), and NMP(30 mL) The resulting solution was stirred at 120° C. Upon completion,the reaction was cooled and added with 30 mL of aqueous NaHCO₃. Theresulting slurry was filtered, rinsed with water, and dried under vacuumat 45° C. to provide4-(2-((4-(3-oxomorpholino)phenyl)amino)pyrimidin-4-yl)benzoic acid(Compound 3) having the below structure:

¹H NMR (400 MHz, DMSO-d₆): δ=13.21 (s, 1H), 9.85 (s, 1H), 8.62 (d, J=5.2Hz, 1H), 8.28 (d, J=8.2 Hz, 2H), 8.10 (d, J=8.2 Hz, 2H), 7.85 (d, J=8.4Hz, 2H), 7.49 (d, J=5.2 Hz, 1H), 7.33 (d, J=9.0 Hz, 2H), 4.19 (s, 2H),3.98 (m, 2H), 3.71 (m, 2H)

To a flask was charged Compound 3 (1.0 g, 2.43 mmol), TBTU (1.0 g, 3.15mmol, 1.3 equiv), glycinamide hydrochloride (0.32 g, 1.2 equiv.), DMSO(9 mL) and i-Pr₂NEt (0.65 g, 2.92 mmol, 1.2 equiv.). Upon reactioncompletion, water (7.7 mL) was added and the resulting slurry wasfiltered and rinsed with DMSO/water (2:1) and water. The isolated solidswere reslurried in 10 mL of MeOH, filtered, washed with MeOH, and driedin a vacuum oven at 45° C. to provideN-(2-amino-2-oxoethyl)-4-(2-((4-(3-oxomorpholino)phenyl)amino)pyrimidin-4-yl)benzamide(Compound 4) having the below structure:

¹H NMR (400 MHz, DMSO-d₆): δ=9.83 (s, 1H), 8.81 (t, J=6.0 Hz, 1H), 8.60(d, J=2.9 Hz, 1H), 8.27 (d, J=8.5 Hz, 2H), 8.05 (d, J=8.4 Hz, 2H), 7.85(d, J=6.9 Hz, 2H), 7.51 (d, J=4.8 Hz, 1H), 7.41 (bs, 1H), 7.32 (d, J=8.9Hz, 2H), 7.06 (bs, 1H), 4.19 (s, 2H), 3.98 (m, 2H), 3.85 (d, J=5.8 Hz,2H), 3.72 (m, 2H); HRMS (ESI+): calcd. for C₂₃H₂₃N₆O₄ [M+H]+: 447.18.found: 447.19.

A suspension of {4-[(cyanomethyl)carbamoyl]phenyl}boronic acid (4.2 g,20.6 mmol), 2,4-dichloropyrimidine (4.3 g, 28.8 mmol), potassiumcarbonate (2.8 kg, 20.6 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (1:1) (84 mg, 0.10 mmol) in acetonitrile (21 mL)and water (11 mL) was sparged with N₂ for 30 minutes. The mixture washeated to 75° C. until the reaction was complete. The mixture was cooledto 60° C. and the layers were separated. An aqueous N-acetyl cysteinesolution (6 mL) was added followed by the addition of water (15 mL). Themixture was cooled to 20° C. The solids were filtered, washed withH₂O/CH₃CN (3:1), and dried at 50° C. to provide4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide having the belowstructure:

¹H NMR (300 MHz, DMSO-d₆): δ 4.36 (d, J=5.5 Hz, 2H), 8.05 (m, J=8.5 Hz,2H), 8.24 (d, J=5.3 Hz, 1H), 8.32 (m, J=8.5 Hz, 2H), 8.89 (d, J=5.2 Hz,1H), 9.39 (t, J=5.5 Hz, 1H). HRMS (ESI+): calcd. for C₁₃H₁₀ClN₄O [M+1]:273.15. found 273.25.

To a flask was charged4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (6.7 g, 24.6 mmol),2-((4-aminophenyl)amino)ethanol (7.5 g, 49.3 mmol, 2.0 equiv), i-Pr₂NEt(4.8 g, 36.9 mmol, 1.5 equiv.), and DMSO (20 mL). The resulting solutionwas stirred at 100° C. Upon reaction completion, the solution was cooledto 20° C. then added onto 135 mL of water. The resulting slurry wasfiltered and rinsed with 70 mL of water. The solids were reslurried ini-PrOH (70 mL). The resulting slurry was filtered and rinsed withi-PrOH. The solids were dried under vacuum and dissolved in 30 mL of THFand heated to 50° C. Water (85 mL) was slowly charged and the slurry wascooled to 20° C. The resulting solids were isolated by filtration,rinsed with THF/water (1:3) and water, and dried at 40° C. to provideN-(cyanomethyl)-4-(2-((4-((2-hydroxyethyl)amino)phenyl)amino)pyrimidin-4-yl)benzamide(Compound 8) having the below structure:

¹H NMR (DMSO-d₆): 9.33 (t, J=5.5 Hz, 1H), 9.24 (s, 1H), 8.48 (d, J=5.1Hz, 1H), 8.24 (d, J=8.4 Hz, 2H), 8.01 (d, J=8.5 Hz, 2H), 7.46 (d, J=8.8Hz, 2H), 7.33 (d, J=5.2 Hz, 1H), 6.58 (d, J=8.9 Hz, 2H), 5.20 (t, J=5.8Hz, 1H), 4.66 (t, J=5.4 Hz, 1H), 4.35 (d, J=5.4 Hz, 2H), 3.57 (q, J=5.8Hz, 2H), 3.08 (q, J=5.8 Hz, 2H); HRMS (ESI+): calcd. for C₂₁H₂₁N₆O₂[M+1]: 389.17. found 389.27.

To a flask was charged4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (4.0 g, 14.7 mmol),phenylenediamine (3.2 g, 29.3 mmol, 2.0 equiv), i-Pr₂NEt (2.9 g, 22.1mmol, 1.5 equiv.), and DMSO (12 mL). The resulting solution was stirredat 60° C. Upon reaction completion, the mixture was cooled to 20° C.then added onto 50 mL of water. The resulting slurry was filtered andrinsed with water followed by i-PrOH. The solids were reslurried ini-PrOH (50 mL), filtered, rinsed with i-PrOH, and dried at 40° C. toafford4-(2-((4-aminophenyl)amino)pyrimidin-4-yl)-N-(cyanomethyl)benzamide(Compound 10) having the below structure:

¹H NMR (400 MHz, DMSO-d₆): δ=9.33 (t, J=5.6 Hz, 1H), 9.21 (s, 1H), 8.47(d, J=5.2 Hz, 1H), 8.24 (d, J=8.5 Hz, 2H), 8.01 (d, J=8.2 Hz, 2H), 7.39(d, J=8.4 Hz, 2H), 7.33 (d, J=5.1 Hz, 1H), 6.56 (d, J=8.4 Hz, 2H), 4.78(bs, 2H), 4.36 (d, J=6.4 Hz, 2H); HRMS (ESI+): calcd. for C₁₉H₁₇N₆O[M+H]: 345.15. found 345.28.

To a flask was charged4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (4.0 g, 14.7 mmol),4′-aminoacetanilide (2.6 g, 17.6 mmol, 1.2 equiv), i-Pr₂NEt (2.9 g, 22.1mmol, 1.5 equiv.), and DMSO (12 mL). The resulting solution was stirredat 120° C. Upon reaction completion, the mixture was cooled to 20° C.and MeOH (30 mL) was slowly added. The resulting slurry was filtered andrinsed with MeOH. The solids were reslurried in 40 mL of MeOH, filtered,rinsed with MeOH, and dried at 40° C. to afford4-(2-((4-acetamidophenyl)amino)pyrimidin-4-yl)-N-(cyanomethyl)benzamide(Compound 12) having the below structure:

¹H NMR (DMSO-d₆): 9.82 (s, 1H), 9.64 (s, 1H), 9.33 (t, J=5.5 Hz, 1H),8.57 (d, J=5.1 Hz, 1H), 8.28 (d, J=8.1 Hz, 2H), (8.04, J=8.5 Hz, 2H),7.72 (d, J=8.9 Hz, 2H), 7.52 (d, J=8.9 Hz, 2H), 7.45 (d, J=5.0 Hz, 1H),4.36 (d, J=5.3 Hz, 2H), 2.03 (s, 3H); HRMS (ESI+): calcd. for C₂₁H₁₉N₆O₂[M+1]: 387.16. found 387.28.

A mixture of 4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (2.0 g,7.2 mmol), 4-(4-aminophenyl)morpholin-3-one (1.4 g, 7.2 mmol) and zincdichloride (98 mg, 0.72 mmol) in N-methylpyrrolidinone (10 mL) wassparged with N₂ for 10 minutes, then heated to 90° C. until the reactionwas deemed complete. The mixture was cooled to 50° C. and water (15 mL)was then slowly added to the reaction mixture. The resulting slurry wascooled to 20° C. and the solids were filtered, rinsed with water anddried. The solids were dissolved in 15 mL of DMSO and heated to 50° C.Methanol (25 mL) was added to the mixture and then cooled to 20° C. Theresulting solids were filtered, rinsed with MeOH, and dried at 60° C.under vacuum to affordN-(cyanomethyl)-4-(2-{[4-(3-oxomorpholin-4-yl)phenyl]amino}pyrimidin-4-yl)benzamide(Compound 13) having the below structure:

¹H NMR (300 MHz, DMSO-d₆): δ 9.83 (s, 1H), 9.34 (t, J=5.5 Hz, 1H), 8.62(d, J=5.2 Hz, 1H), 8.30 (m, J=8.6 Hz, 2H), 8.04 (m, J=8.6 Hz, 2H), 7.85(m, J=8.9 Hz, 2H), 7.51 (d, J=5.2 Hz, 1H), 7.33 (m, J=8.9 Hz, 2H), 4.36(d, J=5.4 Hz, 2H), 4.20 (s, 2H), 3.98 (dd, J=5.9, 4.19 Hz, 2H),3.62-3.79 (m, 2H). HRMS (ESI+): calcd. for C₂₃H₂₁N₆O₃[M+1]: 429.17.found 429.0.

Example 7

This study characterized the effect of CYT-0387 on hepcidin productionin HepG2 cells, a hepatocellular carcinoma cell line. Bone morphogenicproteins (BMPs) is shown to be involved in the transcriptional inductionof hepcidin in hepatocytes by facilitating the association ofconstitutively active Type-II BMP receptor kinases (BMPR-kinase) withType-I BMPR-kinase (Andriopoulos, et al. Nat Genet, 2009. 41(4): p482-7; Zhao, et al., J Clin Invest, 2013. 123(6): p 2337-43). Thisresults in phosphorylation and activation of Type-I BMPR-kinases andsubsequent downstream activation of effector SMAD proteins (SMAD1/5/8)followed by nuclear translocation in association with SMAD4 (Wrana, ColdSpring Harb Perspect Biol, 2013. 5:a011197).

HepG2 cells were preincubated for 2 hours with CYT-0387 (ranging from 0μM to 10 μM) in the presence of 1% FBS then stimulated for 6 hours with10 ng/mL of BMP6. Total RNA was isolated from the cells and analyzed forthe levels of hepcidin by qRT-PCR. GUSB (glucuronidase, beta) was usedas a house keeping control to normalize the levels measured by qRT-PCR.The percentage of hepcidin fold-change induction was calculated (100% isequal to hepcidin induction in vehicle treated cells) and summarized inTable 9. The results showed that CYT-0387 resulted in a dose-dependentinhibition of BMP6-mediated hepcidin induction.

HepG2 cells were preincubated for 2 hours with increasing concentrationsof CYT-0387 (0.02 to 10 M CYT387) in the presence of 1% FBS and thenstimulated for 30 minutes with 10 ng/mL of BMP6. Protein was extractedfrom the lysed cells and analyzed using immunoblot analysis with theantibodies specific for phospho-SMAD1 (Ser463/465), phospho-SMAD5(Ser463/465) and phospho-SMAD8 (Ser465/467) and 3-actin. Rawphospho-SMAD1/5/8 levels were quantified using densitometry software(Image Studio) and normalized to 3-actin levels. The percentage ofphospho-SMAD1/5/8 levels was calculated (100% is equal tophospho-SMAD1/5/8 levels in vehicle treated cells stimulated with 10ng/mL of BMP6) and summarized in Table 9. The results showed thatCYT-0387 resulted in a dose-dependent inhibition of BMP6-mediatedphospho-SMAD1/5/8 levels.

TABLE 9 The normalized percentage of hepcidin fold-change induction andphospho-SMAD1/5/8 levels in HepG2 cells stimulated with BMP6 in thepresence of CYT-0387. hepcidin fold- phospho- CYT-0387 change SMAD1/5/8(μM) induction^(a) SD^(b) levels^(c) SD^(b) 0 100 0 100 0 0.020 115 4083 28 0.039 91 28 80 11 0.078 74 28 91 14 0.156 86 21 96 10 0.313 89 4388 18 0.625 45 24 91 16 1.25 28 21 65 14 2.5 6 13 52 15 5.0 −4 6 40 910.0 −3 4 31 11 ^(a)average induction for n = 2. ^(b)SD: standarddeviation. ^(c)average induction for n = 6.

In addition, biochemical binding assays (DiscoveRx) and in vitro enzymeinhibition assays (LanthaScreen, Life Technologies) were conducted todetermine the binding affinity and inhibition activities of CYT-0387 toType-I BMPR-kinases (ALK2, ALK3, and ALK6) were conducted. Transforminggrowth factor beta receptor 1 (TGFBR1, ALK5) was used as a control todetermine the selectivity to Type-I BMPR-kinases. The results weresummarized in Table 10 and showed that CYT-0387 had higher affinity andinhibitory activities to ALK2 and ALK6 compared to ALK3.

TABLE 10 Biochemical Kd and IC₅₀ values of CYT-0387 to BMPR-kinases. KdIC₅₀ (nM) CYT-0387 (nM) AVG^(a) SD^(b) ALK2 (Acvr1) 25 8 2 ALK3 (BMPR1a)1000 405 131 ALK6 (BMPR1B) 19 107 8 TGFBR1 670 205 37 ^(a)AVG: averagevalue for n = 3. ^(b)SD: standard deviation.

Each of the references including all patents, patent applications andpublications cited in the present application is incorporated herein byreference in its entirety, as if each of them is individuallyincorporated. Further, it would be appreciated that, in the aboveteaching of invention, the skilled in the art could make certain changesor modifications to the invention, and these equivalents would still bewithin the scope of the invention defined by the appended claims of theapplication. Each of the references including all patents, patentapplications and publications cited in the present application isincorporated herein by reference in its entirety, as if each of them isindividually incorporated. Further, it would be appreciated that, in theabove teaching of invention, the skilled in the art could make certainchanges or modifications to the invention, and these equivalents wouldstill be within the scope of the invention defined by the appendedclaims of the application.

What is claimed:
 1. A method of treating myelofibrosis or polycythemiavera comprising administering to a subject in need thereof a compoundselected from the group consisting of:N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II;N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I; andN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III.
 2. The method of claim 1, whereinthe compound is in a crystalline form.
 3. The method of claim 2, whereinthe crystalline form isN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidedihydrochloride monohydrate Form II.
 4. The method of claim 3, whereinthe crystals have unit cell parameters at T=100° K of: a=10.2837(6) Å,b=10.4981(6) Å, c=11.5143(7) Å, α=83.297(2)°, β=87.649(2)°,γ=67.445(2)°, and a triclinic P-1 space group.
 5. The method of claim 3,wherein the crystalline form is characterized by an x-ray powderdiffraction (XRPD) pattern substantially as set forth in FIG.
 5. 6. Themethod of claim 3, wherein the crystalline form is characterized by anx-ray powder diffraction (XRPD) pattern having peaks at about 7.7°,19.3°, 24.0°, 25.7°, and 29.6° 2-θ±0.2° 2-θ.
 7. The method of claim 3,wherein the crystalline form is characterized by differential scanningcalorimetry (DSC) pattern substantially as set forth in FIG.
 8. 8. Themethod of claim 3, wherein the crystalline form is characterized by adynamic vapor sorption (DVS) pattern substantially as set forth in FIG.14.
 9. The method of claim 2, wherein the crystalline form iscrystallineN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form I.
 10. The method of claim 9, whereinthe crystalline form is characterized by an x-ray powder diffraction(XRPD) pattern substantially as set forth in FIG.
 6. 11. The method ofclaim 9, wherein the crystalline form is characterized by an X-raypowder diffraction (XRPD) pattern having peaks at about 13.5°, 20.9°,26.1°, 26.6°, and 28.3° 2-θ±0.2° 2-θ.
 12. The method of claim 9, whereinthe crystalline form is characterized by a differential scanningcalorimetry (DSC) pattern substantially as set forth in FIG.
 9. 13. Themethod of claim 2, wherein the crystalline form is crystallineN-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamidemonohydrochloride anhydrous Form III.
 14. The method of claim 13,wherein the crystalline form is characterized by an x-ray powderdiffraction (XRPD) pattern substantially as set forth in FIG.
 7. 15. Themethod of claim 13, wherein the crystalline form is characterized by anX-ray powder diffraction (XRPD) pattern having peaks at about 12.7°,14.6°, 17.8°, 19.7°, and 23.3° 2-0±0.2° 2-θ.
 16. The method of claim 13,wherein the crystalline form is characterized by a differential scanningcalorimetry (DSC) pattern substantially as set forth in FIG. 10.